CA2133417C - Oil-body protein cis-elements as regulatory signals - Google Patents
Oil-body protein cis-elements as regulatory signals Download PDFInfo
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Abstract
DNA constructs comprising 5' untranslated sequences from genes active from the late globular stage through to embryo maturity are provided. These constructs may be used to obtain expression of a DNA sequence of interest during phases of embry-ogenesis which precede the accumulation of storage proteins.
Description
~,.,1 e~ ~~ ~ ~. ~t WO 93!20216 PCT/CA93/00141 OII~IiODY PROTEIN CIS-ELEMENTS
AS REGULATORY SIGNALS
INTRODUCTION
Technical ~'~eld_ This invention relates to upstream DNA sequences and their use to control expression of genes in developing plant seeds and their use.
Studies in plant gene expression have yielded a number of general conclusions concerning the elements that control expression. PDants, like other organisms both prokaryotic and eukaryotic, contain conserved or consensus sequences upstream (5') of the transcriptional start site of genes which appear capable of regulating transcriptional rates. In eukaryotes, these sequences include a motif found typically about 25 by 5' to the transcriptions! initiation site which has the, sequence TATAAITAA/T and is referred to as a TATA box. The role of this TATA box appears to be to define the transcriptions! start for RNA
polymerase II. A second upstream sequence is referred to as a CART box.
Typically, this is found about.75 bases upstream of the transcriptions! start and is associated with regulating the frequency of transcriptions! initiation. In plants the consensus sequence may be either CCAAT or sometimes AGGA. However, neither of these alternative consensus sequences need be present in all plant genes.
These sequence motifs and their DNA context within 70-90 bases upstream of the transcriptions! start are often referred to as promoters. In general, 5' of the promoter region and most frequently within 200 bases of it are cis-acting elements which confer a variety of properties on the promoter and which can modulate transcrip6onal activity in either a constitutive or a non-constitutive manner. These cis-acting sequences may be referred to as enhancers (if they are responsible for increases in transcription) or silencers (if they are responsible for ~t~BS'Ti'T13TE S~~ET
'~1~~~1'l decreases or suppression of transcription). Enhancers and silencers are frequently the sites at which nuclear proteins bind or interact. 'The modulating nuclear proteins are called traps-acting factors. They are considered to be very important for non-constitutive or regulated expression as they may be the major determinant of the activity of a gene in a particular tissue or organ or in response to an euternal stimulus. The relationship between this protein binding and the enhancer/silencer element may determine the transcriptional activity. The isolation of genes which are activated by heat, light or chemicals such as endogenous hormones or are activated in specific organs such as seeds, leaves or flowers has permitted analysis of factors which may determine how expression is regulated.
In numerous, but not all, cases, it has been shown that the construction of chimeric genes which contain the promoter and optionally cis-elements from a given regulated gee and a coding sequence of a reporter protein not normally associated with that promoter gives rise to regulated expression of the reporter. The use of promoters firom seed-specific genes for the expression of sequences in seed of gages that are either not normally expressed in a seed-specific manner or those that require an altered pattern of expression has been attempted on only a few occasions. In all cases to date, chimeric genes designed for seed-specific ~. ~,e used seed-storage protein regulatory signals and promoters.
However, it is evident from work on storage protein gene expression that expression commences at a fairly late stage in embryogenesis, namely once the embryo has reached (in the case of divots) the classical torpedo shape. Thus, although storage proteins express at high levels and their regulation is often transcriptional, the timing and level of expression may not be ideal for all seed-specific applications. It is, therefore, of interest to identify other seed-specific promoters and enhancers with temporal or cellular specificity different from that of seed storage proteins, such as those from oleosins.
AS REGULATORY SIGNALS
INTRODUCTION
Technical ~'~eld_ This invention relates to upstream DNA sequences and their use to control expression of genes in developing plant seeds and their use.
Studies in plant gene expression have yielded a number of general conclusions concerning the elements that control expression. PDants, like other organisms both prokaryotic and eukaryotic, contain conserved or consensus sequences upstream (5') of the transcriptional start site of genes which appear capable of regulating transcriptional rates. In eukaryotes, these sequences include a motif found typically about 25 by 5' to the transcriptions! initiation site which has the, sequence TATAAITAA/T and is referred to as a TATA box. The role of this TATA box appears to be to define the transcriptions! start for RNA
polymerase II. A second upstream sequence is referred to as a CART box.
Typically, this is found about.75 bases upstream of the transcriptions! start and is associated with regulating the frequency of transcriptions! initiation. In plants the consensus sequence may be either CCAAT or sometimes AGGA. However, neither of these alternative consensus sequences need be present in all plant genes.
These sequence motifs and their DNA context within 70-90 bases upstream of the transcriptions! start are often referred to as promoters. In general, 5' of the promoter region and most frequently within 200 bases of it are cis-acting elements which confer a variety of properties on the promoter and which can modulate transcrip6onal activity in either a constitutive or a non-constitutive manner. These cis-acting sequences may be referred to as enhancers (if they are responsible for increases in transcription) or silencers (if they are responsible for ~t~BS'Ti'T13TE S~~ET
'~1~~~1'l decreases or suppression of transcription). Enhancers and silencers are frequently the sites at which nuclear proteins bind or interact. 'The modulating nuclear proteins are called traps-acting factors. They are considered to be very important for non-constitutive or regulated expression as they may be the major determinant of the activity of a gene in a particular tissue or organ or in response to an euternal stimulus. The relationship between this protein binding and the enhancer/silencer element may determine the transcriptional activity. The isolation of genes which are activated by heat, light or chemicals such as endogenous hormones or are activated in specific organs such as seeds, leaves or flowers has permitted analysis of factors which may determine how expression is regulated.
In numerous, but not all, cases, it has been shown that the construction of chimeric genes which contain the promoter and optionally cis-elements from a given regulated gee and a coding sequence of a reporter protein not normally associated with that promoter gives rise to regulated expression of the reporter. The use of promoters firom seed-specific genes for the expression of sequences in seed of gages that are either not normally expressed in a seed-specific manner or those that require an altered pattern of expression has been attempted on only a few occasions. In all cases to date, chimeric genes designed for seed-specific ~. ~,e used seed-storage protein regulatory signals and promoters.
However, it is evident from work on storage protein gene expression that expression commences at a fairly late stage in embryogenesis, namely once the embryo has reached (in the case of divots) the classical torpedo shape. Thus, although storage proteins express at high levels and their regulation is often transcriptional, the timing and level of expression may not be ideal for all seed-specific applications. It is, therefore, of interest to identify other seed-specific promoters and enhancers with temporal or cellular specificity different from that of seed storage proteins, such as those from oleosins.
3(f The following disclose organ or tissue specific regulatory sequences used to produce tissue or organ-specific expression in transformed plants.
There are several by now "classical" examples of regulated gene expression in non-seed I ~~.~~i ~~1'~
WO X93/30216 ~ PCT/CA93/00141 protein chloramphenicol acetyl transferase could be expressed in a light-regulated and organ-specific manner in transgenic plants if the coding sequence for the reporter protein was fused with the promoter and upstream sequences from a pea gene encoding ribulose bisphosphate carboxylase (Fluhr, Science (1986), x:1106-1112).
Sengupta-Gopaian et al. Proc. Natl. Acad. Sci. USA, (1985) $x:3320-3324 reportod e~cpression of a major storage protein of french beans, called B-phaseolin, in tobacco plants. The gene expressed correctly in the seeds and only at very low levels elsewhere in the plant. However, the constructs used by Sengupta-Gopalan were not chimeric. The entire &phaseolin gene including the native 5'-flanking sequences were used. Subsequent experiments with other species (Radke et al. (1988) Theor. App. Genet. 75:685-694) or other genes (Perez-Grau, L., Goldberg, R.B., 1989, Plant Cell, ,x:1095-1109) showed the fidelity of expression in a seed-specific manner in both Arabidopsis and Brassica.
Itadke et al. (1988), vide supra, used a "tagged" gene i.e., one containing the entire napin gene plus a non-translated "tag".
In tissue and organ specific expression there have been several examples showing that sequences upstream of the transcriptional start may be used to confer tissuelorgan specificity to a gene introduced into plants by genetic engineering. Examples include engineering seed-specific gene regulation (Radke a et al. (1988) vide supra; Bustos et al. (1989), Plant Cell, x:839-853). In both examples, sequences upstream of the coding sequ~ces of seed proteins were linked to a reporter tag (either as RNA or protein) and seed specificity was conferred on expression of the reporter. These were all storage protein genes rather than oleosins. Seed storage proteins have different temporal expression patterns from oleosins.
The DNA motifs that might give rise to seed-specific expression are now the subject of many studies. Marcotte et al. (Marcotte, W.R., Russel, LS., Quantrano, R.S., 1989, Plant Cell, x,:969-97~ studied the Em gene of wheat and proposed two motifs called "Em-boxes" ~rhich might be consensus sequences for seed-specific expression. Interestingly, one of these boxes called EM-2 is similar to that found in other storage protein genes from monocots (triticin-wheat) and WO.93/20216 even divots (B-conglycinin-soybean). Hatzopoulos et al. (1990, Plant Cell, 2_:457-467) investigated the sequences directing embryo-specific expression of a carrot lipid-body protein gene. A number of AT rich motifs were identified, being protected from digestion during DNAse treatment presumably by trans-acting proteins. The motifs identified, however, were not shown to be consensus motifs for other seed-specific genes.
DeClercq et al. Plant Physiol., (1990), 24:970-979 used the promoter of the Arabidopsis 2S albumin and combined coding sequences from both the Arabidopsis and Brazil nut 2S albumins. Fusions were made in regions showing low conserdation. Transformation of both tobacco and Brassica napes y~_c ~pression and correct accumulation of the modified storage proteins. Levels of expression were between 0.05 % and 0.3 % of total cellular protein.
Another example of this form of seed-specific expression of foreign sequences was the expression of lee-enkephalins in seeds. To obtain seed specific expression, a chimeric DNA sequence encoding a 2S albumin and a short oligonucleotide encoding leuphalin (a pentapeptide) was included in the albumin coding sequences between the 6th and 7th cysteines of the native protein (yanderkerhove et al. Bio/Technology, (1989) x:929-932). Again this gene e~cpressed in a seed-specific manner allo~g ~e accumulation of up to 50 nmol lee-enlcaphalin per g of seed.
Genomic clones encoding oil-body proteins with their associated upstream regions have been reported for two species, maize (Zea ways, Bowman-yance and Huang, (1987) J. Biol. Chem., X2_:11275-11279; and Qu and Huang, (1990) J. Biol. Chem., x:2238-2243) and carrot (Hatzopoulos et al. (1990) Plant Cell, x:457-467). cDNAs and genomic clones have also been reported for one cultivated oilseed, Brassaca napes (Murphy, et al. (1991), Biochem. Biophys.
Acts, .1$$:86-94; and Lee and Huang (1991) Plant Physico 96:1395-1397.) .
Reports on the expression of these oil-body protein genes in developing seeds have 3Q varied. In the case of Zea mays, transcription of genes encoding oil-body protein isoforms began quite early in seed development and were easily detected 18 days after pollination. Tn non-endospermic seeds such as the dicotyledonous plant WO 93/20216 ~ ~ ~ ~ ~ ~ ~ PCT/CA93/00141 after pollination. In non-endospermic seeds such as the dicotyledonous plant Brassica napes (Canola), expression of oil-body protein genes seems to occur much later in seed development (Murphy, et al. (1989), Biochem. J., ~$:285-293) than with corn.
There are several by now "classical" examples of regulated gene expression in non-seed I ~~.~~i ~~1'~
WO X93/30216 ~ PCT/CA93/00141 protein chloramphenicol acetyl transferase could be expressed in a light-regulated and organ-specific manner in transgenic plants if the coding sequence for the reporter protein was fused with the promoter and upstream sequences from a pea gene encoding ribulose bisphosphate carboxylase (Fluhr, Science (1986), x:1106-1112).
Sengupta-Gopaian et al. Proc. Natl. Acad. Sci. USA, (1985) $x:3320-3324 reportod e~cpression of a major storage protein of french beans, called B-phaseolin, in tobacco plants. The gene expressed correctly in the seeds and only at very low levels elsewhere in the plant. However, the constructs used by Sengupta-Gopalan were not chimeric. The entire &phaseolin gene including the native 5'-flanking sequences were used. Subsequent experiments with other species (Radke et al. (1988) Theor. App. Genet. 75:685-694) or other genes (Perez-Grau, L., Goldberg, R.B., 1989, Plant Cell, ,x:1095-1109) showed the fidelity of expression in a seed-specific manner in both Arabidopsis and Brassica.
Itadke et al. (1988), vide supra, used a "tagged" gene i.e., one containing the entire napin gene plus a non-translated "tag".
In tissue and organ specific expression there have been several examples showing that sequences upstream of the transcriptional start may be used to confer tissuelorgan specificity to a gene introduced into plants by genetic engineering. Examples include engineering seed-specific gene regulation (Radke a et al. (1988) vide supra; Bustos et al. (1989), Plant Cell, x:839-853). In both examples, sequences upstream of the coding sequ~ces of seed proteins were linked to a reporter tag (either as RNA or protein) and seed specificity was conferred on expression of the reporter. These were all storage protein genes rather than oleosins. Seed storage proteins have different temporal expression patterns from oleosins.
The DNA motifs that might give rise to seed-specific expression are now the subject of many studies. Marcotte et al. (Marcotte, W.R., Russel, LS., Quantrano, R.S., 1989, Plant Cell, x,:969-97~ studied the Em gene of wheat and proposed two motifs called "Em-boxes" ~rhich might be consensus sequences for seed-specific expression. Interestingly, one of these boxes called EM-2 is similar to that found in other storage protein genes from monocots (triticin-wheat) and WO.93/20216 even divots (B-conglycinin-soybean). Hatzopoulos et al. (1990, Plant Cell, 2_:457-467) investigated the sequences directing embryo-specific expression of a carrot lipid-body protein gene. A number of AT rich motifs were identified, being protected from digestion during DNAse treatment presumably by trans-acting proteins. The motifs identified, however, were not shown to be consensus motifs for other seed-specific genes.
DeClercq et al. Plant Physiol., (1990), 24:970-979 used the promoter of the Arabidopsis 2S albumin and combined coding sequences from both the Arabidopsis and Brazil nut 2S albumins. Fusions were made in regions showing low conserdation. Transformation of both tobacco and Brassica napes y~_c ~pression and correct accumulation of the modified storage proteins. Levels of expression were between 0.05 % and 0.3 % of total cellular protein.
Another example of this form of seed-specific expression of foreign sequences was the expression of lee-enkephalins in seeds. To obtain seed specific expression, a chimeric DNA sequence encoding a 2S albumin and a short oligonucleotide encoding leuphalin (a pentapeptide) was included in the albumin coding sequences between the 6th and 7th cysteines of the native protein (yanderkerhove et al. Bio/Technology, (1989) x:929-932). Again this gene e~cpressed in a seed-specific manner allo~g ~e accumulation of up to 50 nmol lee-enlcaphalin per g of seed.
Genomic clones encoding oil-body proteins with their associated upstream regions have been reported for two species, maize (Zea ways, Bowman-yance and Huang, (1987) J. Biol. Chem., X2_:11275-11279; and Qu and Huang, (1990) J. Biol. Chem., x:2238-2243) and carrot (Hatzopoulos et al. (1990) Plant Cell, x:457-467). cDNAs and genomic clones have also been reported for one cultivated oilseed, Brassaca napes (Murphy, et al. (1991), Biochem. Biophys.
Acts, .1$$:86-94; and Lee and Huang (1991) Plant Physico 96:1395-1397.) .
Reports on the expression of these oil-body protein genes in developing seeds have 3Q varied. In the case of Zea mays, transcription of genes encoding oil-body protein isoforms began quite early in seed development and were easily detected 18 days after pollination. Tn non-endospermic seeds such as the dicotyledonous plant WO 93/20216 ~ ~ ~ ~ ~ ~ ~ PCT/CA93/00141 after pollination. In non-endospermic seeds such as the dicotyledonous plant Brassica napes (Canola), expression of oil-body protein genes seems to occur much later in seed development (Murphy, et al. (1989), Biochem. J., ~$:285-293) than with corn.
Methods and compositions are described for the exploitation of an oil-body protein transcriptional regulatory sequence and optionally its accompanying 5' untranslated leader sequence for the expression of heterologous genes in a seed-specific manner. The method includes the steps of transforming a plant cell with a DNA construct comprising the regulatory sequence and a DNA
sequence other than the open reading frame native to the regulatory sequence, generating a plant from the transformed cell and growing it under conditions whereby seed is produced and the DNA sequence is expressed under the transcriptional control of the regulatory region. These sequences will be valuable in applications where expression of a seed-borne product neais to be modified, enhanced or suppressed. They could also be used to produce modified seeds containing foreign proteins to increase the intrinsic value of the seed.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows a schematic diagram of vector pPAW4 enclosing an oleosin regulatory sequence, an initiation colon, foreign DNA to be expressed, an oleosin terminator s~uence and an ampicillin resistance gene.
Fig. 2A shows the DNA sequence of an Arabifopsis genomic clone encoding a l8KDa oil-body protein. The open reading frame is interrupted by a short intron (which is marked) and the two axons are translated and indicated in IUPAC single letter amino-acid codes.
Fig. 2B shows the restriction fragment from an Agrobacterium EMBL3 genomic library which encloses the Arabidopsis l8KDa oil-body protein coding sequence. The approximate position of the coding region is highlighted.
Fig. 3 shows the effects of lO~cM ABA on the developmental expression of oleosin mRNA using a Northern blot analysis of total RNA. A) W4 93/Z0216 ' PCT/CA93/00141 (?O~eg per lane) using 50 ng32P dCTP labelled OB990 as a probe (spec. act. 109 dpm/~cg DNA). Heart-(H): (13-day), torpedo-('I~ (17-day), and cotyledonary-(C) (21-25 day) stage microsponr-derived embryos with (+) and without (-) treatment for 48 h with 10~,M ABA. The blot was exposed to Kodak XARS film at 70°C
for 20 minutes. The apparent size difference of the mRNAs in the different lanes is due to interfering quantities of starch in the different mRNA preparations.
All tlu lanes wart equally loaded as judged by OD260 measurements and EtBr-staining. B) A 4.5 hour exposure of Fig. 3-A C) Relative intensity of the mRNA
accumulation as determined by scanning densitometry.
Fig. 4 shows the tissue specificity of oleosin. 50 ~cg of poly (A)+
RNA of mots (R), callus (Ca), Cotyledons (Co), leaves (L), and 24-day post-anthesis zygotic embryos (E) was probed with 50 ng of 3zP dCTP labelled OB990 (spec. act. 1f' dpml~cg DNA).
Fig. 5 shows tl~ developmental sensitivity of oil body protein synthesis to applied ABA. An estimated 10,000 dpm were loaded per well for paged samples of controls (lanes A,C,E,G) and ABA treated (lanes B,D,F,H).
All samples were for 2d with ABA, the labeled for 4 h with 1.85 MBq/mL ~S]methionine. Lanes A and B, 10-d-old cultures, sieved on 62~cm s to obtain globular embryos. Lanes C and D, 13-d-old ~culturPs sieved on 125~m screens to obtain heart stage embryos. Lanes E and F, 17-d-old cultures sieved on 250~cm screens to obtain torpedo to early cotyledonary embryos.
Lanes 'G and H, 25-d-old cultures, sieved on 500~cm screens to obtain cotyledonary stage embryos.
~JfEF DESCRIPTION OF THE SPECIFIC EMBODIMENTS
In accordance with the subject invention, DNA constructs are provided which allow for modulation of plant phenotype in seed, particularly during early phases of embryogenesis. The DNA constructs provide for regulation of transcription in sped; using 5' untranslated sequences from genes active from the late globular stage through to embryo maturity (cotyledonary stage).
Dawhstream from and under t<anscriptional initiation-regulation of an oil body protein gene initiation region will be a DNA sequence of interest which will v ,',. v.~ ~ ) f.r..~c)J~~,~
sequence other than the open reading frame native to the regulatory sequence, generating a plant from the transformed cell and growing it under conditions whereby seed is produced and the DNA sequence is expressed under the transcriptional control of the regulatory region. These sequences will be valuable in applications where expression of a seed-borne product neais to be modified, enhanced or suppressed. They could also be used to produce modified seeds containing foreign proteins to increase the intrinsic value of the seed.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows a schematic diagram of vector pPAW4 enclosing an oleosin regulatory sequence, an initiation colon, foreign DNA to be expressed, an oleosin terminator s~uence and an ampicillin resistance gene.
Fig. 2A shows the DNA sequence of an Arabifopsis genomic clone encoding a l8KDa oil-body protein. The open reading frame is interrupted by a short intron (which is marked) and the two axons are translated and indicated in IUPAC single letter amino-acid codes.
Fig. 2B shows the restriction fragment from an Agrobacterium EMBL3 genomic library which encloses the Arabidopsis l8KDa oil-body protein coding sequence. The approximate position of the coding region is highlighted.
Fig. 3 shows the effects of lO~cM ABA on the developmental expression of oleosin mRNA using a Northern blot analysis of total RNA. A) W4 93/Z0216 ' PCT/CA93/00141 (?O~eg per lane) using 50 ng32P dCTP labelled OB990 as a probe (spec. act. 109 dpm/~cg DNA). Heart-(H): (13-day), torpedo-('I~ (17-day), and cotyledonary-(C) (21-25 day) stage microsponr-derived embryos with (+) and without (-) treatment for 48 h with 10~,M ABA. The blot was exposed to Kodak XARS film at 70°C
for 20 minutes. The apparent size difference of the mRNAs in the different lanes is due to interfering quantities of starch in the different mRNA preparations.
All tlu lanes wart equally loaded as judged by OD260 measurements and EtBr-staining. B) A 4.5 hour exposure of Fig. 3-A C) Relative intensity of the mRNA
accumulation as determined by scanning densitometry.
Fig. 4 shows the tissue specificity of oleosin. 50 ~cg of poly (A)+
RNA of mots (R), callus (Ca), Cotyledons (Co), leaves (L), and 24-day post-anthesis zygotic embryos (E) was probed with 50 ng of 3zP dCTP labelled OB990 (spec. act. 1f' dpml~cg DNA).
Fig. 5 shows tl~ developmental sensitivity of oil body protein synthesis to applied ABA. An estimated 10,000 dpm were loaded per well for paged samples of controls (lanes A,C,E,G) and ABA treated (lanes B,D,F,H).
All samples were for 2d with ABA, the labeled for 4 h with 1.85 MBq/mL ~S]methionine. Lanes A and B, 10-d-old cultures, sieved on 62~cm s to obtain globular embryos. Lanes C and D, 13-d-old ~culturPs sieved on 125~m screens to obtain heart stage embryos. Lanes E and F, 17-d-old cultures sieved on 250~cm screens to obtain torpedo to early cotyledonary embryos.
Lanes 'G and H, 25-d-old cultures, sieved on 500~cm screens to obtain cotyledonary stage embryos.
~JfEF DESCRIPTION OF THE SPECIFIC EMBODIMENTS
In accordance with the subject invention, DNA constructs are provided which allow for modulation of plant phenotype in seed, particularly during early phases of embryogenesis. The DNA constructs provide for regulation of transcription in sped; using 5' untranslated sequences from genes active from the late globular stage through to embryo maturity (cotyledonary stage).
Dawhstream from and under t<anscriptional initiation-regulation of an oil body protein gene initiation region will be a DNA sequence of interest which will v ,',. v.~ ~ ) f.r..~c)J~~,~
be prepared which allow for integration of the transcription cassette into the genome of a plant cell. Conveniently, a multiple cloning site downstream from the seed specific transcriptional initiation region may be included so that the integration construct may be employed for a variety of DNA sequences in an S efficient manner.
Of particular interest is a regulatory sequence from an oil body protein gene, preferably an oil body protein gene expressed in dicotyledonous oil seeds. It has been reported that oil-body proteins accumulate considerably later than either oils (triacylglycerides) or storage proteins. This later expression would limit the value of any promoters associated with these genes for seed-specific a~pressio~n as they could not be used for modification of expression of genes during early phases of embryogenesis. Surprisingly, however, expression of these genes in dicotyledonous oilseeds was found to occur much earlier than had hitherto been believed. Thus, the promoters and upstream elements of these genes are valuable for a variety of uses invol ring the modification of metabolism during phases of embryogwhich precede the accumulation of stoiage proteins.
Oil~ody proteins have been identified in a wide range of ta~wnomically diverse spocies (see, for example, Moreau et al: Plant Physiol.
(1980), ~:1I76-1180; Qu ~ al. Biochem. J:, (1986) x:57-6~). These proteins are uniquely localized in oil-bodies and are not found in organelles of vegetative f tissues. In Brassica »apus (rapeseed) there are at least three polypeptides associated with the oil-bodies of developing seeds (Taylor et al. (1990), Planter, 1$x,:18-26). The numbers and sizes of oil-body associated proteins may vary from species to species. In corn, for example, there are four immunologically distinct ~ polypeptides found in oil-bodies (Bowman-Vance and liuang, 1988, J. Biol.
Chem., ~:1476-1481). Oleosins have been shown to comprise regions of alternate hydrophilicity, hydrophobicity and hydrophilicity (Bowman-Vance and Huang, 1987, J. Biol. Chem., X2_:11275-11279). The amino acid sequences of oleosins from corn, rid and carrot have been obtained. See Qu and Huang, 1990, J. Biol. Cfum., x:2238-2243; Hatzopoulos ex al. 1990, Plant Cell; ~:457-467; respectively. In an oilseed such as rapeseed, oleosin may comprise between 896 (Taylor et al. 1990, Planter, x$1":18-26) and 20% (Murphy et al. 1989, W093/20216 ~ ~ ~ ~ ~ ~ ' PCT/CA93/OOldl Biochem. 1., ?x$:285-293) of total seed protein. Such a level is comparable to that found for many seed storage proteins.
Of particular interest is a transcriptional initiation region associated with early embryogenesis, particularly the period preceding expression of storage proteins, so that in the early development of seed, it provides the desired level of transcription of the DNA sequence of interest. Normal plant embryogenesis typically goes through a series of defined phases. For dicotyledonous seeds, embryogenesis includes the following phases: globular stage, heart stage, torpedo stage, and cotyledonary stage. For the purposes of this application, the definition of these terms is provided by Ray, Steves, and Fultz in Botany, (Saunders College Publishing), Chapter 17, page 294. Normally, the transcriptional initiation region will be obtainable from a gene which is expressed in the early formation of seed.
Desirably the transcriptional initiation region maintains its activity from the late globular through cotyledonary stage, more desirably continues active from the globular stage through the heart, torpedo and cotyledonary stages of embryog~esis. By obtainable is intended a transcriptional initiation region having a nucleotide sequence-sufficie~ntly similar to that of a natural oil body protein gene transcriptional initiation region sequence to provide for transcription in the early formation of seed. The sequence may be naturally occurring, sjmthetic or partially ZO synthetic.
The transcriptional initiation region from the oil body protein generally will be provided in a which will include in the 5'-3' direction of transcription, a transcriptional initiation region, a DNA sequence of interest and a transcriptional termination region, wherein the transcriptional regulatory regions are operably joined and functional in plant cells. One or more introns may also be present. After each manipulation, a DNA to be used in the final construct may be restricted and operably joined to other DNA to be used in the final construct, where each of the partial constructs may be cloned in the same or different plasmids. In a preferred embodiment, a coding sequ~ce with a compatible restziction site may be ligated at the position con~esponding to colon ~1 of the oil-body protein gene. A schematic diagram of this substitution is shown in figure 1.
The recombinant coding sequence may be inserted in such a way that it completely <~~~~4i~
Of particular interest is a regulatory sequence from an oil body protein gene, preferably an oil body protein gene expressed in dicotyledonous oil seeds. It has been reported that oil-body proteins accumulate considerably later than either oils (triacylglycerides) or storage proteins. This later expression would limit the value of any promoters associated with these genes for seed-specific a~pressio~n as they could not be used for modification of expression of genes during early phases of embryogenesis. Surprisingly, however, expression of these genes in dicotyledonous oilseeds was found to occur much earlier than had hitherto been believed. Thus, the promoters and upstream elements of these genes are valuable for a variety of uses invol ring the modification of metabolism during phases of embryogwhich precede the accumulation of stoiage proteins.
Oil~ody proteins have been identified in a wide range of ta~wnomically diverse spocies (see, for example, Moreau et al: Plant Physiol.
(1980), ~:1I76-1180; Qu ~ al. Biochem. J:, (1986) x:57-6~). These proteins are uniquely localized in oil-bodies and are not found in organelles of vegetative f tissues. In Brassica »apus (rapeseed) there are at least three polypeptides associated with the oil-bodies of developing seeds (Taylor et al. (1990), Planter, 1$x,:18-26). The numbers and sizes of oil-body associated proteins may vary from species to species. In corn, for example, there are four immunologically distinct ~ polypeptides found in oil-bodies (Bowman-Vance and liuang, 1988, J. Biol.
Chem., ~:1476-1481). Oleosins have been shown to comprise regions of alternate hydrophilicity, hydrophobicity and hydrophilicity (Bowman-Vance and Huang, 1987, J. Biol. Chem., X2_:11275-11279). The amino acid sequences of oleosins from corn, rid and carrot have been obtained. See Qu and Huang, 1990, J. Biol. Cfum., x:2238-2243; Hatzopoulos ex al. 1990, Plant Cell; ~:457-467; respectively. In an oilseed such as rapeseed, oleosin may comprise between 896 (Taylor et al. 1990, Planter, x$1":18-26) and 20% (Murphy et al. 1989, W093/20216 ~ ~ ~ ~ ~ ~ ' PCT/CA93/OOldl Biochem. 1., ?x$:285-293) of total seed protein. Such a level is comparable to that found for many seed storage proteins.
Of particular interest is a transcriptional initiation region associated with early embryogenesis, particularly the period preceding expression of storage proteins, so that in the early development of seed, it provides the desired level of transcription of the DNA sequence of interest. Normal plant embryogenesis typically goes through a series of defined phases. For dicotyledonous seeds, embryogenesis includes the following phases: globular stage, heart stage, torpedo stage, and cotyledonary stage. For the purposes of this application, the definition of these terms is provided by Ray, Steves, and Fultz in Botany, (Saunders College Publishing), Chapter 17, page 294. Normally, the transcriptional initiation region will be obtainable from a gene which is expressed in the early formation of seed.
Desirably the transcriptional initiation region maintains its activity from the late globular through cotyledonary stage, more desirably continues active from the globular stage through the heart, torpedo and cotyledonary stages of embryog~esis. By obtainable is intended a transcriptional initiation region having a nucleotide sequence-sufficie~ntly similar to that of a natural oil body protein gene transcriptional initiation region sequence to provide for transcription in the early formation of seed. The sequence may be naturally occurring, sjmthetic or partially ZO synthetic.
The transcriptional initiation region from the oil body protein generally will be provided in a which will include in the 5'-3' direction of transcription, a transcriptional initiation region, a DNA sequence of interest and a transcriptional termination region, wherein the transcriptional regulatory regions are operably joined and functional in plant cells. One or more introns may also be present. After each manipulation, a DNA to be used in the final construct may be restricted and operably joined to other DNA to be used in the final construct, where each of the partial constructs may be cloned in the same or different plasmids. In a preferred embodiment, a coding sequ~ce with a compatible restziction site may be ligated at the position con~esponding to colon ~1 of the oil-body protein gene. A schematic diagram of this substitution is shown in figure 1.
The recombinant coding sequence may be inserted in such a way that it completely <~~~~4i~
replaces the coding sequence of the oil-body protein gene and is thus flanked at its 3' end by the oil-body protein gene terminator and polyadenylation signal.
Alternatively, polymerise chain reaction amplification may be carried out to produce DNA fragments containing the transcriptional initiation region conveniently flanked by restriction sites. The amplified fragments can be joined to the casing sequence for a polypeptide of interest, in a transcriptional or translational fusion, for example, to produce a chimeric gene in which the coding sequence of the polypeptide of interest is transcribed under the control of the transcription initiation region on the PCR amplified fragment.
The transcriptional initiation region may be native to or homologous to the host cell, or foreign or heterologous to the host cell. By foreign is intended that the transcriptional initiation region is not found in the wild-type host into which the construct comprising the transcriptional initiation region is inserted.
Generally, the regulatory .sequence comprises DNA of up to 1.5 Kb 5' of the translational start of an oil-body protein gene. lfiis sequence may be modified at the position corresponding to the first colon of the desired protein by sito-directed mutagenesis (Kunkel TA, 1985, Proc. Natl. Acid. Sci. USA, $x:488-492) or by introduction of a convenient linker oligonucleotide by ligation if a suitable resuiction site is found near the N-terminal codan.
In some cases it will be desirable to express the DNA sequence of interest as a fusion protein, particularly as a fusion protein with the oil body protein. The DNA sequence of interest can be insert~l by routine techniques into the oil body protein coding sequence, in frdrne with the oil body protein coding sequence, such that transcription of the chimeric gene will produce a fusion protein. The fusion protein will preferably contain the coding region for amino acids number 44 through 122 in the Arabidopsis oil body protein as shown an Figure 2A, or the ~uivalent region from an oil body protein of a skies other than Ar~abadopsis, to provide for transport to the oil body in cases where this is desirable.
In order to isolate oil body protein coding sequences from other species, at least two approaches may be used. The first is to use the Arabidopsis clone described in the Examples as a probe in genomic libraries of other plant ' , ., , WO 93/20216 ~' ~ '~ '~ ~ ~ ~ PCT/CA93/00141 species. This clone will hybridize well with oleosin clones from closely related species, in particular, essentially all cruciferous plants. For species which are evolutionarily divergent from Arabidopsis, for example, solanaceae, leguminaceae and all monocotyledons, an alternative method involves the use of an antibody 5 raised against the gene product of an oleosin clone such as the Arabidopsis clone.
This antibody may be used to scan a seed-derived cDNA expression library, for example using lambda gtll; Huynh et al. (1985) in cDNA Cloning, Vol. 1, A
Practical Approach, Ed. Grover IRI. Press, pp. 49-78. This approach yields a cDNA clone of the oleosin for the new species which may then be used to isolate 10 the genomic clone from a genomic library of that species by standard DNA
hybridization techniques.
The DNA sequence of interest may be any open reading frame encoding a peptide of interest, for example, an enzyme, or a sequence complementary to a genomic sequence, where the genomic sequence may be at least one of an open reading frame, an intron, a nun-coding leader sequence, or any other sequence where the complementary sequence will inhibit transcription, messenger RNA processing, for example splicing or translation. The DNA
sequence of interest may be synthetic, naturally derived or a combination thereof.
Depending upon the nature of the DNA sequence of interest, it~may be desirable to synthesize the sequence with plant preferr~ colons. The plant preferred a colons may be determined from the colons of highest frequency in the proteins expre$sed in the largest amount in the particular plant species of interest.
The DNA sequence of interest may encode any of a variety of recombinant proteins. Examples of recombinant proteins which might be expressed by this procedure include anticoagulants, such as Hirudin, lympholdnes such as those of the interleukin family, peptide hormones such as gonadotrophin releasing hormone, immunologic~l reagents such as multi or single-chain antibodies and a variety of industrial valuable enzymes such as proteases, lipases and polyglucan hydrolases.
3Q The termination region which is employed will be primarily one of convenience, since the termination regions appear to be relatively interchangeable.
The termination region may be native with the DNA sequence of interest, or may ~~.~~~~1'l be derived from another source. Convenient termination regions are available and include the 3' end of the oil body protein gene terminator and polyadenylation signal from the same gene from which the 5' regulatory region is obtained.
Alternatively, a different terminator and polyadenylation signal may be employed with similar results, for example, the terminator of the nopaline synthase gene of ~lgrobacterium.
The expression cassette may additionally contain a means for identifying transformed cells and/or selecting for transformed cells. For example the recombinant gene may be linked with a constitutively expressed selectable marlxr such as a gene for antibiotic resistance or herbicide resistance or a screarable marker, such as a gene conferring bioluminescence or colored properties to transformed cells.
The DNA sequence of interest flanked at its 5' end by the oil-body protein promoter and regulatory sequences and at its 3' end by a terminator may be introduced into a suitable transformation vector including Agrobacterium Ti or binary plasmids, or a simple cloning plasmid (e.g., pUCl9, pBR322) for use in diroct DNA uptake to plant cells via microinjection, electroporation, PEG-mediated uptake or a biolistic method. These methods are well known to those stilled in the art of plant transformation. See, for example, I~oisch et al.
(1985), ZO Science, 227:1229-1231; Newhaus and Spangenberg (1990). Physiol. Plant, 79:213 217; and Sandford et al. (1990), Physiol. Plant, 79:206-209.
Transforms plants may be obtained from the transformed cells using standard regeneration protocols (see for example: Moloney et al. (1989), Plant Cell Rep., $:238-242) compatible with the transfornation method.
The expression cassette, constructed as described above, expresses essentially preferentially in developing seeds. The plant cells which have been transformed with an appropriate fusion peptide therefore are grown into plants in accordance with conv~tional ways and allowed to set seed. See, for example, McCormick et al., Plant Cell Rep. (1986) 5:81-84. Two or more generations may be groom and either pollinated with the same transformed strain or different strains, identifying the resulting hybrid having the desired phenotypic characteristic, to ensure that the subject phenotypic characteristic is stably maintained and inherited and then seeds harvested for isolation of the peptide of interest or for use to provide seeds with the new phenotypic property. The regenerated plants are then cultivated identically to non-recombinant plants in growth chambers, greenhouses or in the field and will show seed-specific S expression of the recombinant gene at the ml2NA level and often at the level of polypeptide or protein.
It is possible that the polypeptide/protein will itself be valuable and could be extracted and, if desired, further purified. Alternatively the polypeptidelprotein or even the mRNA itself may be used to confer a new biochemical phenotype upon the developing seed. New phenotypes could include such modiFtcations as altered seed-protein or seed oil composition, enhanced production of pre-existing desirable products or properties and the reduction or even suppression of an undesirable gene product using antisense, ribozyme or co-suppression technologies (Izant and Weintraub (1984), Cell 36: 1007-1015, IS antisense; Hazeihoff and Gerlach (1988), Nature 334:585-591, ribozyme;
Napoli, et al. {1990), Plant Cell, 2:279-289, co-suppression).
If the transformation has been performed to produce a new seed protein or peptide which requires extraction, this can be done using aqueous extraction with or without low concentrations of detergents, such as non-denaturing amounts of sodium dodecyl sulphate (;SDS), TritonTM-X-100, TweenTM
20, iviEGA-8 or any other detergent known not co irreversibly inactivate the desired protein. To extract the protein or polypeptide, dry seeds are ground by hand or in a mechanical grinder to produce an aqueous slurry or suspension. This can be resolved into three phases (particulate, aqueous soluble, and hydrophobic) by centrifugation, such as at 50,000 x g. Depending upon the nature of the product, it may be further purified in each of these phases and after solublization, may be selectively precipitated by the use of ammonium sulfate or puriF~ed using column chromatography, for example, using ion exchange, gel filtrates or affinity matrices.
While the ideal host for the regulatory sequence reported here would be a cruciferous plant, it is possible to use these promoters in a wide variety of plant species given the relatively high conservation oleosin of genes. The major b, ~r ~.~~~lv W0.93/20216 PCT/CA93/00141 barrier to the use of these promoters is between monocotyledonous and dicotyledonous species. For transformations involving this specific expression on a monocot, a monocot olesin regulatory sequence should be used. For divot seed-specific expression, a divot oleosin regulatory sequence should be employed.
The reported sequence can be used in a wide variety of dicotyledonous plants, including all members of the Brassica genus and crucifers in general.
Solanaceous plants, such as tobacco and tomato, also recognize the sequences and show correct regulation of expression in developing seeds.
It is expected that the desired proteins would be expressed in all embryonic tissue, although different cellular e~cpression can be detected in different tissues of the embryonic auis and cotyledons. This invention has a variety of uses which include improving the intrinsic value of plant seeds by their accumulation of altered polypeptides or novel recombinant peptides or by the incorporation or elimination of a metabolic step. In its simplest embodiment, use of this invention IS may result in improved protein quality (for example, increased concentrations of essential or rare amino acids), improved liquid quality by a modifs~.~.tion of fatty acid composition, or improved or elevated carbohydrate composition. P~camples include the expression of sulfur-rich proteins, such as those found in lupins or brazil nuts in a seed deficient in sulphurous amino acid residues' Alternatively, a fatty aryl coenzyme A (COA) a transferase enzyme capable of modifying fatty Los in triglycerides (storage lipid) could be expressed. In cases where a recombinant pmtein is allows to acxumulate in the seed, the protein could also be a peptide which has pharmaceutical, industrial or nutritional value. In this rise, the peptide could be extracts from the seed and used in crude or purified form, as appropriate for the intended use. The protein could be one truly foreign to the plant kingdom, such as an animal hormone, enzyme, lymphokine, anticoagulant, or the like could be expressed in seed. The heterologous protein could then be extracted from the seeds and used for experimental, nutritional or pharmaceutical purposes after partial or complete purification.
The following examples are offered by way of illustration and not by limitation.
W0.93/20216 ~ ~ ~ ~ ~~ 1 rt PCT/CA93/0(1141 SAMPLES
The oil body protein gene from Arabidopsis was isolated on a lSkb insert present in a clone from an Arabidopsis thaliana v. Columbia genomic library in phage ~ EMBL3A by hybridization to a B. napes oleosin clone. A
l.8kb fragment containing approximately 868 base pairs 5' of the oleosin protein translational start was subcloned into a plasmid vector. The Arabidopsis ~ 8 KDa oleosin gene is conveniently cloned as a 1803 by fragment flanked by Ncol and Kpnl sites in a vector called pPAVV4 (see Figure 1). In order to convert the fragment into an expression cassette for general use with a variety of foreign/alternative genes, two modifications must be made. Firstly, using the technique of site-directed mutagenesis (Kunkel, supra) mutations at positions -2, -1 and +4 are introduced using a mis-matched oligonucleotide. The mutations required are A to T (-2), A to C (-1) and G to A (+4). These mutations have the . effect of creating a BspHl site at positions -2 to +4. The BspHl site (T/CATGA) encloses the ATG initiation colon and gives a recessed end compatible with an Ncol cut. A second modification involves digestion with EcoRV and P~iscl which releases a 658 by fragment containing most of the coding sequence of the native oleosin. This leaves blunt ends at the cut sites which on separation of the vector and an ancillary sequence from the EcoRV-Idfscl fragment, permits recircularization of the vector-promoter terminator combination. This recircularization is performed in the presence of an oligonucleotide linker containing restriction sites not found in the original 1803 Kb fragment.
Gn recircularization, a plasmid containing all the upstream sequences of the oleosin gene, a transcriptional start site and an initiation colon embedded in a BspHl site is obtained. Thirty-one bases downstream of this is a short polylinker containing one or more unique restriction sites. To introduce any DNA sequence into this cassette the foreign sequence should have, or should be modified to contain, a BspHl or Ncol site at the initial ATG position. For sequences to be expmessed as proteins this will assure conservation of the distance between the "cap" site and the initiator colon.
iw ~. ~) tl ~ .,~. '~
W0.93/20216 PCT/CA93/00141 The DNA sequence to be inserted should terminate with a cohesive ~d of a restriction site not found on the plasmid. The polylinker interposed into the e~cpression cassette may be chosen with this site in mind. Digesting the plasmid with BspHl and the appropriate restriction enzyme for the 3' end of the 5 foreign sequence will ensure that a directional cloning of the desired DNA
fragment may be effected. Using appropriate ligation conditions, the plasmid acpre~on cassette with BspHl and a site compatible with the desired DNA, fragment are incubated together to produce a ligated product as shown in Figure 1.
The complete construct from Ncol-Kpnl is now excised and 10 introduood into an appropriate plant transformation vector such as an Agritan plasmid. In order to introduce the construct into common Agrobacteriwn plasmids such as Bin 19 (Bevan, Nucl. Acid Research (1984) x:8711-8721) it may be necessary to use one of the additional restriction sites in p>asmid pPAW4. In one scenario the plasmid could be cut with Smal and Kpnl.
Alternatively, polymerise chain reaction amplification may be carried out to produce DNA fragments containing the transcriptional initiation region conveniently flanked by restriction sites. The amplified fragments can be joined to the casing sequence for a polypeptide of interest, in a transcriptional or translational fusion, for example, to produce a chimeric gene in which the coding sequence of the polypeptide of interest is transcribed under the control of the transcription initiation region on the PCR amplified fragment.
The transcriptional initiation region may be native to or homologous to the host cell, or foreign or heterologous to the host cell. By foreign is intended that the transcriptional initiation region is not found in the wild-type host into which the construct comprising the transcriptional initiation region is inserted.
Generally, the regulatory .sequence comprises DNA of up to 1.5 Kb 5' of the translational start of an oil-body protein gene. lfiis sequence may be modified at the position corresponding to the first colon of the desired protein by sito-directed mutagenesis (Kunkel TA, 1985, Proc. Natl. Acid. Sci. USA, $x:488-492) or by introduction of a convenient linker oligonucleotide by ligation if a suitable resuiction site is found near the N-terminal codan.
In some cases it will be desirable to express the DNA sequence of interest as a fusion protein, particularly as a fusion protein with the oil body protein. The DNA sequence of interest can be insert~l by routine techniques into the oil body protein coding sequence, in frdrne with the oil body protein coding sequence, such that transcription of the chimeric gene will produce a fusion protein. The fusion protein will preferably contain the coding region for amino acids number 44 through 122 in the Arabidopsis oil body protein as shown an Figure 2A, or the ~uivalent region from an oil body protein of a skies other than Ar~abadopsis, to provide for transport to the oil body in cases where this is desirable.
In order to isolate oil body protein coding sequences from other species, at least two approaches may be used. The first is to use the Arabidopsis clone described in the Examples as a probe in genomic libraries of other plant ' , ., , WO 93/20216 ~' ~ '~ '~ ~ ~ ~ PCT/CA93/00141 species. This clone will hybridize well with oleosin clones from closely related species, in particular, essentially all cruciferous plants. For species which are evolutionarily divergent from Arabidopsis, for example, solanaceae, leguminaceae and all monocotyledons, an alternative method involves the use of an antibody 5 raised against the gene product of an oleosin clone such as the Arabidopsis clone.
This antibody may be used to scan a seed-derived cDNA expression library, for example using lambda gtll; Huynh et al. (1985) in cDNA Cloning, Vol. 1, A
Practical Approach, Ed. Grover IRI. Press, pp. 49-78. This approach yields a cDNA clone of the oleosin for the new species which may then be used to isolate 10 the genomic clone from a genomic library of that species by standard DNA
hybridization techniques.
The DNA sequence of interest may be any open reading frame encoding a peptide of interest, for example, an enzyme, or a sequence complementary to a genomic sequence, where the genomic sequence may be at least one of an open reading frame, an intron, a nun-coding leader sequence, or any other sequence where the complementary sequence will inhibit transcription, messenger RNA processing, for example splicing or translation. The DNA
sequence of interest may be synthetic, naturally derived or a combination thereof.
Depending upon the nature of the DNA sequence of interest, it~may be desirable to synthesize the sequence with plant preferr~ colons. The plant preferred a colons may be determined from the colons of highest frequency in the proteins expre$sed in the largest amount in the particular plant species of interest.
The DNA sequence of interest may encode any of a variety of recombinant proteins. Examples of recombinant proteins which might be expressed by this procedure include anticoagulants, such as Hirudin, lympholdnes such as those of the interleukin family, peptide hormones such as gonadotrophin releasing hormone, immunologic~l reagents such as multi or single-chain antibodies and a variety of industrial valuable enzymes such as proteases, lipases and polyglucan hydrolases.
3Q The termination region which is employed will be primarily one of convenience, since the termination regions appear to be relatively interchangeable.
The termination region may be native with the DNA sequence of interest, or may ~~.~~~~1'l be derived from another source. Convenient termination regions are available and include the 3' end of the oil body protein gene terminator and polyadenylation signal from the same gene from which the 5' regulatory region is obtained.
Alternatively, a different terminator and polyadenylation signal may be employed with similar results, for example, the terminator of the nopaline synthase gene of ~lgrobacterium.
The expression cassette may additionally contain a means for identifying transformed cells and/or selecting for transformed cells. For example the recombinant gene may be linked with a constitutively expressed selectable marlxr such as a gene for antibiotic resistance or herbicide resistance or a screarable marker, such as a gene conferring bioluminescence or colored properties to transformed cells.
The DNA sequence of interest flanked at its 5' end by the oil-body protein promoter and regulatory sequences and at its 3' end by a terminator may be introduced into a suitable transformation vector including Agrobacterium Ti or binary plasmids, or a simple cloning plasmid (e.g., pUCl9, pBR322) for use in diroct DNA uptake to plant cells via microinjection, electroporation, PEG-mediated uptake or a biolistic method. These methods are well known to those stilled in the art of plant transformation. See, for example, I~oisch et al.
(1985), ZO Science, 227:1229-1231; Newhaus and Spangenberg (1990). Physiol. Plant, 79:213 217; and Sandford et al. (1990), Physiol. Plant, 79:206-209.
Transforms plants may be obtained from the transformed cells using standard regeneration protocols (see for example: Moloney et al. (1989), Plant Cell Rep., $:238-242) compatible with the transfornation method.
The expression cassette, constructed as described above, expresses essentially preferentially in developing seeds. The plant cells which have been transformed with an appropriate fusion peptide therefore are grown into plants in accordance with conv~tional ways and allowed to set seed. See, for example, McCormick et al., Plant Cell Rep. (1986) 5:81-84. Two or more generations may be groom and either pollinated with the same transformed strain or different strains, identifying the resulting hybrid having the desired phenotypic characteristic, to ensure that the subject phenotypic characteristic is stably maintained and inherited and then seeds harvested for isolation of the peptide of interest or for use to provide seeds with the new phenotypic property. The regenerated plants are then cultivated identically to non-recombinant plants in growth chambers, greenhouses or in the field and will show seed-specific S expression of the recombinant gene at the ml2NA level and often at the level of polypeptide or protein.
It is possible that the polypeptide/protein will itself be valuable and could be extracted and, if desired, further purified. Alternatively the polypeptidelprotein or even the mRNA itself may be used to confer a new biochemical phenotype upon the developing seed. New phenotypes could include such modiFtcations as altered seed-protein or seed oil composition, enhanced production of pre-existing desirable products or properties and the reduction or even suppression of an undesirable gene product using antisense, ribozyme or co-suppression technologies (Izant and Weintraub (1984), Cell 36: 1007-1015, IS antisense; Hazeihoff and Gerlach (1988), Nature 334:585-591, ribozyme;
Napoli, et al. {1990), Plant Cell, 2:279-289, co-suppression).
If the transformation has been performed to produce a new seed protein or peptide which requires extraction, this can be done using aqueous extraction with or without low concentrations of detergents, such as non-denaturing amounts of sodium dodecyl sulphate (;SDS), TritonTM-X-100, TweenTM
20, iviEGA-8 or any other detergent known not co irreversibly inactivate the desired protein. To extract the protein or polypeptide, dry seeds are ground by hand or in a mechanical grinder to produce an aqueous slurry or suspension. This can be resolved into three phases (particulate, aqueous soluble, and hydrophobic) by centrifugation, such as at 50,000 x g. Depending upon the nature of the product, it may be further purified in each of these phases and after solublization, may be selectively precipitated by the use of ammonium sulfate or puriF~ed using column chromatography, for example, using ion exchange, gel filtrates or affinity matrices.
While the ideal host for the regulatory sequence reported here would be a cruciferous plant, it is possible to use these promoters in a wide variety of plant species given the relatively high conservation oleosin of genes. The major b, ~r ~.~~~lv W0.93/20216 PCT/CA93/00141 barrier to the use of these promoters is between monocotyledonous and dicotyledonous species. For transformations involving this specific expression on a monocot, a monocot olesin regulatory sequence should be used. For divot seed-specific expression, a divot oleosin regulatory sequence should be employed.
The reported sequence can be used in a wide variety of dicotyledonous plants, including all members of the Brassica genus and crucifers in general.
Solanaceous plants, such as tobacco and tomato, also recognize the sequences and show correct regulation of expression in developing seeds.
It is expected that the desired proteins would be expressed in all embryonic tissue, although different cellular e~cpression can be detected in different tissues of the embryonic auis and cotyledons. This invention has a variety of uses which include improving the intrinsic value of plant seeds by their accumulation of altered polypeptides or novel recombinant peptides or by the incorporation or elimination of a metabolic step. In its simplest embodiment, use of this invention IS may result in improved protein quality (for example, increased concentrations of essential or rare amino acids), improved liquid quality by a modifs~.~.tion of fatty acid composition, or improved or elevated carbohydrate composition. P~camples include the expression of sulfur-rich proteins, such as those found in lupins or brazil nuts in a seed deficient in sulphurous amino acid residues' Alternatively, a fatty aryl coenzyme A (COA) a transferase enzyme capable of modifying fatty Los in triglycerides (storage lipid) could be expressed. In cases where a recombinant pmtein is allows to acxumulate in the seed, the protein could also be a peptide which has pharmaceutical, industrial or nutritional value. In this rise, the peptide could be extracts from the seed and used in crude or purified form, as appropriate for the intended use. The protein could be one truly foreign to the plant kingdom, such as an animal hormone, enzyme, lymphokine, anticoagulant, or the like could be expressed in seed. The heterologous protein could then be extracted from the seeds and used for experimental, nutritional or pharmaceutical purposes after partial or complete purification.
The following examples are offered by way of illustration and not by limitation.
W0.93/20216 ~ ~ ~ ~ ~~ 1 rt PCT/CA93/0(1141 SAMPLES
The oil body protein gene from Arabidopsis was isolated on a lSkb insert present in a clone from an Arabidopsis thaliana v. Columbia genomic library in phage ~ EMBL3A by hybridization to a B. napes oleosin clone. A
l.8kb fragment containing approximately 868 base pairs 5' of the oleosin protein translational start was subcloned into a plasmid vector. The Arabidopsis ~ 8 KDa oleosin gene is conveniently cloned as a 1803 by fragment flanked by Ncol and Kpnl sites in a vector called pPAVV4 (see Figure 1). In order to convert the fragment into an expression cassette for general use with a variety of foreign/alternative genes, two modifications must be made. Firstly, using the technique of site-directed mutagenesis (Kunkel, supra) mutations at positions -2, -1 and +4 are introduced using a mis-matched oligonucleotide. The mutations required are A to T (-2), A to C (-1) and G to A (+4). These mutations have the . effect of creating a BspHl site at positions -2 to +4. The BspHl site (T/CATGA) encloses the ATG initiation colon and gives a recessed end compatible with an Ncol cut. A second modification involves digestion with EcoRV and P~iscl which releases a 658 by fragment containing most of the coding sequence of the native oleosin. This leaves blunt ends at the cut sites which on separation of the vector and an ancillary sequence from the EcoRV-Idfscl fragment, permits recircularization of the vector-promoter terminator combination. This recircularization is performed in the presence of an oligonucleotide linker containing restriction sites not found in the original 1803 Kb fragment.
Gn recircularization, a plasmid containing all the upstream sequences of the oleosin gene, a transcriptional start site and an initiation colon embedded in a BspHl site is obtained. Thirty-one bases downstream of this is a short polylinker containing one or more unique restriction sites. To introduce any DNA sequence into this cassette the foreign sequence should have, or should be modified to contain, a BspHl or Ncol site at the initial ATG position. For sequences to be expmessed as proteins this will assure conservation of the distance between the "cap" site and the initiator colon.
iw ~. ~) tl ~ .,~. '~
W0.93/20216 PCT/CA93/00141 The DNA sequence to be inserted should terminate with a cohesive ~d of a restriction site not found on the plasmid. The polylinker interposed into the e~cpression cassette may be chosen with this site in mind. Digesting the plasmid with BspHl and the appropriate restriction enzyme for the 3' end of the 5 foreign sequence will ensure that a directional cloning of the desired DNA
fragment may be effected. Using appropriate ligation conditions, the plasmid acpre~on cassette with BspHl and a site compatible with the desired DNA, fragment are incubated together to produce a ligated product as shown in Figure 1.
The complete construct from Ncol-Kpnl is now excised and 10 introduood into an appropriate plant transformation vector such as an Agritan plasmid. In order to introduce the construct into common Agrobacteriwn plasmids such as Bin 19 (Bevan, Nucl. Acid Research (1984) x:8711-8721) it may be necessary to use one of the additional restriction sites in p>asmid pPAW4. In one scenario the plasmid could be cut with Smal and Kpnl.
15 The rr,~ulting purified fragment then is ligated to a Kpnl oligonucleotide linker and digested withy Kpnl. This provides a non-directional Kpnl fragment for i~oduction into Bin 19. Alternatively, the construct may be excised with Kpnl and BamHl and ligated directionally into pBIN 19 pnwiously cut with the same re~riction enzymes. The resulting Agrobacterirrm binary plasmid is mobilized into a disarmed Agrobacterium strain by tripartite mating (Ditta, et al. (1980), PNAS
77: 7347 7351) or DNA transformation of competent Agrobacteriwn (An, (1988), Plant Mol. Biology Manual, A3 1-19, Kluwer Academic, Dordrecht, Netherlands).
The Agnvbacterium harboring the recombinant Bin 19 is used to transform any susceptible plant, e.g., Brassica sp. by standard explant co-cultivation (Horsch et al. (198, supra). The transformed cells are selected in culture with kanamycin using the co-transferral antibiotic resistance genes (neomycin phosphotransferase) also contained between the T-DNA borders of pain 19. These transformed calls are induced to regenerate whole plants by standard procedures (e.g. for an oilseed such as rapeseed: ~, Molameyr et al. Plant Cell Rep., (1989), $: 238-242). Tht regenerated plants are permitted to flower and are self fertilized (or may be cross-fertilized). In cases wrhere the foreign DNA
in the construct encodes a translatable product, this product may be isolated from 16 ~~ ~ ~ j ,~ {~ ~ ~ ~ PGT/GA93/00141 aqueous extractions of the mature seed and subsequent fractionation of the slurry by centrifugation (30 min at 100,000 xg). Depending on the desired product it may partition with any one of the three phases obtained. It may be localized in the pellet, aqueous soluble phase or in the lipid film on the surface of the centrifuged sample.
Alternatively, it may not be necessary to extract the product as the purpose of the expression may be to divert metabolism in the seed thus changing the phenotype of the seed (e.g. by altering size or colour of the seed, changing the ratio of fatty acid residues in the seed or interdicting a particular metabolic step considered to render the seed less useful or valuable. Such metabolic steps might ir~lude the production of antinutritional secondary products which reduce the value or desirability of the seed when present. In such cases, the seed, per se, is simply harvested and used in accordance with usual procedures.
A number of constructs containing varying amounts of the DNA
from the 5' transcriptional initiation region of the Arabidopsis oleosin gee joined operably to the coding region for ~-glucuronidase (GUS) were prepared using PCR. The constructs are designated according ~'o the amount of the oleosin 5' region contained, for example, the 25~ construct has approximately f 2500 base pairs of the.oleosin 5' region. The constructs were introduced into Brassica napes and tobacco and the expression of the ~-glucuronidase gene was measured as described in detail below. The GUS expression results of five constructs, the 2500, the 1200, the 800, the 600 and the 200 constructs in transformed Brassica napes plants are shown in Table I. A negative control (untransformed plant) is also shown. The GUS expression results of two constructs, the 2500 and the 800 constructs, in transformed tobacco plants are shown in Table II. Table III shows the developmental timing of the expression of the oleosin promoter in transgenic embryos.
3Q The constructs were made using standard molecular biology techniques, including restriction enzyme digestion, ligation and polymerase chain W0.93/20216 PCT/CA93/00141 reaction (PCR). As an illustration of the techniques employed, the construction of the 800 construct is described in detail.
In order to obtain a DNA fragment containing approximately 800 base pairs from the 5' transcriptional initiation region of the Arabidopsis oleosin gene in a configuration suitable for ligation to a GUS coding sequence, a PCR
based approach was used. This involves the use of the polymerise chain reaction to amplify the procise sequence desired for the expression analysis. To perform the necessary PCR amplification, two oligonucleotide primers were synthesized (Milligen-Biosearch, Cyclone DNA synthesizer) having the following sequences:
Pst 1 oltosin seq 5' primer: 5'CACTG(~IGGAAGTGTGTGGTAA 3' (GVR10) IS The italicized bases correspond to nucleotide positions -833 to -817 in the sequence reported in Fig. 2A. The additional nucleotides 5' of this sequence in the primer are not identical to the oleosin gene, but were included in order to place a Pstl site at the 5' end of the amplification product. The Pstl site ~, is tmderlined.
A second (3') primer was synthesized which had the following sequence:
3' primer (Aa,P 1) oleosin seq 5-CTA~~~ATCCTGT?TAGTAGAGAGAAT~3 Smal This primer contains the precise complement (shown in italics) to the sequence reported in Fig. 2A from base -13 to -30. In addition, it contains a 13 bases at the 5' end. lfiis sequence is not complementary to the oleosin gene, but was added to provide two (overlapping) restriction sites, Smal and BamHl, at the 3' end of the amplification pmduct to facilitate cloning of the PCR
fragment.
;~~. -~'~'~r7 !'..;.~' 1 .. .2JW a'.o ~ ~~~ 7 x -r.::- .l ., t... . k..' .,. c, : rrr-.. , . :,~ > , s . "r.5~ , _ . .. . ~. ... ..~....s ..
r..,..~.~........m:~r. T...,rw,%'r.~_~:/~?F71~83~ ~m , ....".,~,...,a , ,__ ... ,.,... .:.., i'. -x ...... .... ~,.....~~1.. ... ..... _, , .. , r , ~..
~:.. a , . ". .. . v. , ... , . , . .. . ~ . .. ... . .r. ... , ..
77: 7347 7351) or DNA transformation of competent Agrobacteriwn (An, (1988), Plant Mol. Biology Manual, A3 1-19, Kluwer Academic, Dordrecht, Netherlands).
The Agnvbacterium harboring the recombinant Bin 19 is used to transform any susceptible plant, e.g., Brassica sp. by standard explant co-cultivation (Horsch et al. (198, supra). The transformed cells are selected in culture with kanamycin using the co-transferral antibiotic resistance genes (neomycin phosphotransferase) also contained between the T-DNA borders of pain 19. These transformed calls are induced to regenerate whole plants by standard procedures (e.g. for an oilseed such as rapeseed: ~, Molameyr et al. Plant Cell Rep., (1989), $: 238-242). Tht regenerated plants are permitted to flower and are self fertilized (or may be cross-fertilized). In cases wrhere the foreign DNA
in the construct encodes a translatable product, this product may be isolated from 16 ~~ ~ ~ j ,~ {~ ~ ~ ~ PGT/GA93/00141 aqueous extractions of the mature seed and subsequent fractionation of the slurry by centrifugation (30 min at 100,000 xg). Depending on the desired product it may partition with any one of the three phases obtained. It may be localized in the pellet, aqueous soluble phase or in the lipid film on the surface of the centrifuged sample.
Alternatively, it may not be necessary to extract the product as the purpose of the expression may be to divert metabolism in the seed thus changing the phenotype of the seed (e.g. by altering size or colour of the seed, changing the ratio of fatty acid residues in the seed or interdicting a particular metabolic step considered to render the seed less useful or valuable. Such metabolic steps might ir~lude the production of antinutritional secondary products which reduce the value or desirability of the seed when present. In such cases, the seed, per se, is simply harvested and used in accordance with usual procedures.
A number of constructs containing varying amounts of the DNA
from the 5' transcriptional initiation region of the Arabidopsis oleosin gee joined operably to the coding region for ~-glucuronidase (GUS) were prepared using PCR. The constructs are designated according ~'o the amount of the oleosin 5' region contained, for example, the 25~ construct has approximately f 2500 base pairs of the.oleosin 5' region. The constructs were introduced into Brassica napes and tobacco and the expression of the ~-glucuronidase gene was measured as described in detail below. The GUS expression results of five constructs, the 2500, the 1200, the 800, the 600 and the 200 constructs in transformed Brassica napes plants are shown in Table I. A negative control (untransformed plant) is also shown. The GUS expression results of two constructs, the 2500 and the 800 constructs, in transformed tobacco plants are shown in Table II. Table III shows the developmental timing of the expression of the oleosin promoter in transgenic embryos.
3Q The constructs were made using standard molecular biology techniques, including restriction enzyme digestion, ligation and polymerase chain W0.93/20216 PCT/CA93/00141 reaction (PCR). As an illustration of the techniques employed, the construction of the 800 construct is described in detail.
In order to obtain a DNA fragment containing approximately 800 base pairs from the 5' transcriptional initiation region of the Arabidopsis oleosin gene in a configuration suitable for ligation to a GUS coding sequence, a PCR
based approach was used. This involves the use of the polymerise chain reaction to amplify the procise sequence desired for the expression analysis. To perform the necessary PCR amplification, two oligonucleotide primers were synthesized (Milligen-Biosearch, Cyclone DNA synthesizer) having the following sequences:
Pst 1 oltosin seq 5' primer: 5'CACTG(~IGGAAGTGTGTGGTAA 3' (GVR10) IS The italicized bases correspond to nucleotide positions -833 to -817 in the sequence reported in Fig. 2A. The additional nucleotides 5' of this sequence in the primer are not identical to the oleosin gene, but were included in order to place a Pstl site at the 5' end of the amplification product. The Pstl site ~, is tmderlined.
A second (3') primer was synthesized which had the following sequence:
3' primer (Aa,P 1) oleosin seq 5-CTA~~~ATCCTGT?TAGTAGAGAGAAT~3 Smal This primer contains the precise complement (shown in italics) to the sequence reported in Fig. 2A from base -13 to -30. In addition, it contains a 13 bases at the 5' end. lfiis sequence is not complementary to the oleosin gene, but was added to provide two (overlapping) restriction sites, Smal and BamHl, at the 3' end of the amplification pmduct to facilitate cloning of the PCR
fragment.
;~~. -~'~'~r7 !'..;.~' 1 .. .2JW a'.o ~ ~~~ 7 x -r.::- .l ., t... . k..' .,. c, : rrr-.. , . :,~ > , s . "r.5~ , _ . .. . ~. ... ..~....s ..
r..,..~.~........m:~r. T...,rw,%'r.~_~:/~?F71~83~ ~m , ....".,~,...,a , ,__ ... ,.,... .:.., i'. -x ...... .... ~,.....~~1.. ... ..... _, , .. , r , ~..
~:.. a , . ". .. . v. , ... , . , . .. . ~ . .. ... . .r. ... , ..
These two primers were used in a PCR amplification reaction to produce DNA fragment containing the sequence between nucleotides -833 and -13 of the oleosin gene with a Pstl site at the 5' end and Smal and BamHl sites at the 3' end. PCR amplification ,uas performed using the enzyme TaqTM enzyme (Perkin-Elmer-Cetus) using the conditions recommended by the enzyme manufacturer and a temperature program of 92°C (denaturation) 1 min, 55°C
(annealing) 1 min and 72°C (elongation) 1 min. The template was the oleosin genomic clone shown in Figure 28, top panel, which in the original 7. library isolate contained approximately I5 kilobases of Arabidop.sis DNA.
The amplification product (OLEO p800) was gel purified on 0.7%
agarose, recovered using the glass bead method of Vogelstein and Gillespie (Preparative and analytical purification of DNA from agarose. Proc. Natl.
Acad.
5ci. USA 1979 '76:615-619) and digested with Pstl. The digestion product was gel purified and end filled using DNA polymerase Klenow fragment then cut with Smal to produce a blunt ended fragment. This was cloned into the Smal site of pUC 19 to yield the plasmid pUC OLEOp800. Using the asymmetric positioning of the Accl site in the insert (at the position corresponding to -649 in the oleosin gene as shown in Figure 2B) it was possible to select both orientations of insertion into pUC vector. The clone having the insert oriented sur.l~ that the 5' most end of the amplified fragment (in the direction of transcription) is proximal to the unique Hind III site in the puCl9 cloning vector and the 3' most end of the amplified fragment is proximal to the unique I;~:o RI site in the pUCl9 closing vector.
The resulting plasmid was then cut with BarnHl to yield the fragment OLEOp800 flanked by BamH 1 sites. 'This fragment, BamH 1-OLE0800, was cloned into the BamHl sites of a BamHl digested plasmid designated HspGUS 1559. HspGUS 1559 is a plasmid used as a binary vector in Agrobacterium, derived from the vector pCGN 1559 (htacl3ride and Summerfeldt, 1990, Plant Molecular Biology, 14, 269-2?6) with an insert containing heat shock promoter (flanked by BamHl sites), the ~i-glucuranidase open reading frame and a nopaline synthase terminator (derived from pB1221, Jefferson RA in Cloning Vectors 1988, Eds. Pouwels P., Enger-Valk BE, Brammer WJ., Elsevier Science WO 93/20216 .. i. t ~ ~~ PCT/CA93/00141 Pub BV, Amsterdam section VII, Ail l). BamHl digestion of HspGUS1559 results in the release of the heat shock promoter and permits the insertion of any other BamHl fragment in its place. The BamHl-OLEOp800 fragment was ligated into this site to yield the Agrobacterium pOLEOp800GUS1559. This plasmid was used to transform E. coli and the amplified plasmid was introduced into Agrobacterium (strain EHA101) by electroporation as described above (Rogers et al., 1988, Plant Molecular Biology Manual, A2: 1-12, Eds. Gelvin S. and Schilperoort, R. Kluwer Academic, Dordrecht, Netherlands).
The resultant Agrobacterium strain (EHA 101 x pOLF.Op800GUS1559) was used to transform Brassica napes plants by the method of Moloney et al. (Moloney, M.M., Walker, J.M., Sharma, K.K. (1989) Plant Cell Reports 8:238-242) or tobacco plants by the method of Horsch et al.
(Horsch et al. Science (1985) 227:1299-1302). The resultant transgenic plants were allowed to set seed, and GUS expression assays (Jefferson R.A. (1987), Plant Mol. Biol. Rep. 5 387-405) were performed on the developing seeds and also on non-reproductive plant parts as controls. GUS expression reported is an average obtained from approximately five seeds from each of approximately five different transgenic plants.
The other constructs were prepared by the same SCR method described above using the appropriate primers for amplifying the -2500 fragment, the -1200 fragment, the -600 fragment or the -200 fragment. The results in Brassica napes expressed as specific activity of GUS enzyme are shown in Table I. The results in tobacco are shown in Table II.
These results demonstrate that the oleosin fragment from -833 to -813 used in the 800 construct contains sufficient information to direct seed-spa;ific expression of a reporter gene in transgenic Brassica napes embryos as early as heart stage and that the Arobidopsis oleosin promoter is capable of directing transcription in plants other than Arabidopsis. These experiments also show that the sequences present in this promoter construct contain the cis elements required 34 for an increase in transcription in response to the addition of abscisic acid, a characteristic of the native oleosin promoter.
WO 93/20216 ~ ~_ ~ ~ ~ ~, ~ PCT/CA93/00141 It should be noted that the seed-specific expression demonstrated here does not depend on interactions with the native terminator of an oleosin gene 3' end. In this example, the 3' oleosin terminator was replaced by a terminator derived from the nopaline synthase gene of Agrobacteriarm. Thus, the sequence in 5 the 800 construct is suf~~cient to drive the desired expression profile independent of ancillary sequences.
Y
1 r r' r,~'~k'~
~t~.:... . . '~'~'J a ... .Vine., ~. h.1 -~4.~.._. '~. ~..v.. .-t.W... :'"v~ ~
., m fi r WU~ 93/Z0216 PCT/CA93/00141 Table I
~~p~~~~pressiQn in .8rassica napes .
$ GUS Activity (in pmol product/min/mg protein) Promoter/GUS
Construct ABA* -ABA
2500 10,185 7,709 444 46.9 88.2 11,607 1200 18,298 1,795 8,980 800 2,256 475 285 277 650 7,130 600 1,506 144 1,365 200 18.1 64.8 260 5.9 26 11 Negative 18.4 13.9 300 6.1 30 14 r Control-Non-transform~l Plant *ABA is treatment for abscisicacid 24 hours with 10'sM prior to GUS
activity measurement Seed Root ~f ~gm Seed (Late-(Toraedo Stage) ~Q~yledon~
._::: . ~ . ..._. .: ,.... , . :.
W0.93/20216 PCT/CA93/00141 Table II
S~~ific Expression in Tobacco S Promoter/GUS GUS Activity (in pmol productlmin/mg protein) Constcvcts Mature Seeds 2500 11,330 800 10,970 Table III
~eve~mental Expression in Br~~sica na~us GUS ACTIVITY (in pmol product/min/mg protein)' Promoter/ Early Mid- Late r GUS Heart Torpedo CotyledonaryCotyledonaryCotyleaonary 2500 272 1207 2541 1819 11,607 1200 124 262 388 5094 8,980 800 149 260 962 2617 7,128 600 59 41 29 38 1,365 Negative 11 14 14 Comrol The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from tine spirit or scope of the appended claims.
(annealing) 1 min and 72°C (elongation) 1 min. The template was the oleosin genomic clone shown in Figure 28, top panel, which in the original 7. library isolate contained approximately I5 kilobases of Arabidop.sis DNA.
The amplification product (OLEO p800) was gel purified on 0.7%
agarose, recovered using the glass bead method of Vogelstein and Gillespie (Preparative and analytical purification of DNA from agarose. Proc. Natl.
Acad.
5ci. USA 1979 '76:615-619) and digested with Pstl. The digestion product was gel purified and end filled using DNA polymerase Klenow fragment then cut with Smal to produce a blunt ended fragment. This was cloned into the Smal site of pUC 19 to yield the plasmid pUC OLEOp800. Using the asymmetric positioning of the Accl site in the insert (at the position corresponding to -649 in the oleosin gene as shown in Figure 2B) it was possible to select both orientations of insertion into pUC vector. The clone having the insert oriented sur.l~ that the 5' most end of the amplified fragment (in the direction of transcription) is proximal to the unique Hind III site in the puCl9 cloning vector and the 3' most end of the amplified fragment is proximal to the unique I;~:o RI site in the pUCl9 closing vector.
The resulting plasmid was then cut with BarnHl to yield the fragment OLEOp800 flanked by BamH 1 sites. 'This fragment, BamH 1-OLE0800, was cloned into the BamHl sites of a BamHl digested plasmid designated HspGUS 1559. HspGUS 1559 is a plasmid used as a binary vector in Agrobacterium, derived from the vector pCGN 1559 (htacl3ride and Summerfeldt, 1990, Plant Molecular Biology, 14, 269-2?6) with an insert containing heat shock promoter (flanked by BamHl sites), the ~i-glucuranidase open reading frame and a nopaline synthase terminator (derived from pB1221, Jefferson RA in Cloning Vectors 1988, Eds. Pouwels P., Enger-Valk BE, Brammer WJ., Elsevier Science WO 93/20216 .. i. t ~ ~~ PCT/CA93/00141 Pub BV, Amsterdam section VII, Ail l). BamHl digestion of HspGUS1559 results in the release of the heat shock promoter and permits the insertion of any other BamHl fragment in its place. The BamHl-OLEOp800 fragment was ligated into this site to yield the Agrobacterium pOLEOp800GUS1559. This plasmid was used to transform E. coli and the amplified plasmid was introduced into Agrobacterium (strain EHA101) by electroporation as described above (Rogers et al., 1988, Plant Molecular Biology Manual, A2: 1-12, Eds. Gelvin S. and Schilperoort, R. Kluwer Academic, Dordrecht, Netherlands).
The resultant Agrobacterium strain (EHA 101 x pOLF.Op800GUS1559) was used to transform Brassica napes plants by the method of Moloney et al. (Moloney, M.M., Walker, J.M., Sharma, K.K. (1989) Plant Cell Reports 8:238-242) or tobacco plants by the method of Horsch et al.
(Horsch et al. Science (1985) 227:1299-1302). The resultant transgenic plants were allowed to set seed, and GUS expression assays (Jefferson R.A. (1987), Plant Mol. Biol. Rep. 5 387-405) were performed on the developing seeds and also on non-reproductive plant parts as controls. GUS expression reported is an average obtained from approximately five seeds from each of approximately five different transgenic plants.
The other constructs were prepared by the same SCR method described above using the appropriate primers for amplifying the -2500 fragment, the -1200 fragment, the -600 fragment or the -200 fragment. The results in Brassica napes expressed as specific activity of GUS enzyme are shown in Table I. The results in tobacco are shown in Table II.
These results demonstrate that the oleosin fragment from -833 to -813 used in the 800 construct contains sufficient information to direct seed-spa;ific expression of a reporter gene in transgenic Brassica napes embryos as early as heart stage and that the Arobidopsis oleosin promoter is capable of directing transcription in plants other than Arabidopsis. These experiments also show that the sequences present in this promoter construct contain the cis elements required 34 for an increase in transcription in response to the addition of abscisic acid, a characteristic of the native oleosin promoter.
WO 93/20216 ~ ~_ ~ ~ ~ ~, ~ PCT/CA93/00141 It should be noted that the seed-specific expression demonstrated here does not depend on interactions with the native terminator of an oleosin gene 3' end. In this example, the 3' oleosin terminator was replaced by a terminator derived from the nopaline synthase gene of Agrobacteriarm. Thus, the sequence in 5 the 800 construct is suf~~cient to drive the desired expression profile independent of ancillary sequences.
Y
1 r r' r,~'~k'~
~t~.:... . . '~'~'J a ... .Vine., ~. h.1 -~4.~.._. '~. ~..v.. .-t.W... :'"v~ ~
., m fi r WU~ 93/Z0216 PCT/CA93/00141 Table I
~~p~~~~pressiQn in .8rassica napes .
$ GUS Activity (in pmol product/min/mg protein) Promoter/GUS
Construct ABA* -ABA
2500 10,185 7,709 444 46.9 88.2 11,607 1200 18,298 1,795 8,980 800 2,256 475 285 277 650 7,130 600 1,506 144 1,365 200 18.1 64.8 260 5.9 26 11 Negative 18.4 13.9 300 6.1 30 14 r Control-Non-transform~l Plant *ABA is treatment for abscisicacid 24 hours with 10'sM prior to GUS
activity measurement Seed Root ~f ~gm Seed (Late-(Toraedo Stage) ~Q~yledon~
._::: . ~ . ..._. .: ,.... , . :.
W0.93/20216 PCT/CA93/00141 Table II
S~~ific Expression in Tobacco S Promoter/GUS GUS Activity (in pmol productlmin/mg protein) Constcvcts Mature Seeds 2500 11,330 800 10,970 Table III
~eve~mental Expression in Br~~sica na~us GUS ACTIVITY (in pmol product/min/mg protein)' Promoter/ Early Mid- Late r GUS Heart Torpedo CotyledonaryCotyledonaryCotyleaonary 2500 272 1207 2541 1819 11,607 1200 124 262 388 5094 8,980 800 149 260 962 2617 7,128 600 59 41 29 38 1,365 Negative 11 14 14 Comrol The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from tine spirit or scope of the appended claims.
Claims (33)
1. A method of expressing a DNA sequence of interest in a seed cell, said method comprising:
(a) transforming a plant capable of developing seed with an expression cassette comprising in the 5'-3' direction of transcription as operably linked components, a transcriptional regulatory region obtained from an oil body protein gene, a DNA sequence of interest heterologous to said regulatory region wherein said heterologous DNA sequence does not encode a plant acyl-ACP thioesterase or .beta.-ketoacyl-ACP synthetase II, and a transcriptional termination region; and (b) growing said plant under conditions whereby seed is produced in which said DNA sequence is expressed under transcriptional control of said regulatory region.
(a) transforming a plant capable of developing seed with an expression cassette comprising in the 5'-3' direction of transcription as operably linked components, a transcriptional regulatory region obtained from an oil body protein gene, a DNA sequence of interest heterologous to said regulatory region wherein said heterologous DNA sequence does not encode a plant acyl-ACP thioesterase or .beta.-ketoacyl-ACP synthetase II, and a transcriptional termination region; and (b) growing said plant under conditions whereby seed is produced in which said DNA sequence is expressed under transcriptional control of said regulatory region.
2. A method of expressing a DNA sequence of interest in a seed cell, said method comprising:
(a) transforming a plant capable of developing seed with an expression cassette comprising in the 5'-3' direction of transcription as operably linked components, a transcriptional regulatory region obtained from an Arabidopsis oil body protein gene, a DNA sequence of interest heterologous to said regulatory region, and a transcriptional termination region; and (b) growing said plant under conditions whereby seed is produced in which said DNA sequence is expressed under transcriptional control of said regulatory region.
(a) transforming a plant capable of developing seed with an expression cassette comprising in the 5'-3' direction of transcription as operably linked components, a transcriptional regulatory region obtained from an Arabidopsis oil body protein gene, a DNA sequence of interest heterologous to said regulatory region, and a transcriptional termination region; and (b) growing said plant under conditions whereby seed is produced in which said DNA sequence is expressed under transcriptional control of said regulatory region.
3. The method according to claim 1 or 2, wherein said oil body protein gene is expressed during a phase of embryogenesis which precedes accumulation of storage proteins.
4. The method according to claim 3 wherein said phase is from the formation of a globular embryo through to early cotyledonary stage.
5. The method according to claim 1, wherein said oil body protein gene is from a dicotyledenous plant.
6. The method according to claim 3, wherein said phase is selected from the group consisting of globular, heart, torpedo and cotyledonary.
7. The method according to claim 5, wherein said dicotyledonous plant is Brassica napus or Arabidopsis.
8. The method according to claim 1 or 2, wherein said plant is other than Arabidopsis.
9. A DNA construct comprising:
a chimeric gene comprising (a) a transcriptional regulatory region obtained from an Arabidopsis oil body protein gene fused to (b) a DNA
sequence of interest heterologous to said regulatory region.
a chimeric gene comprising (a) a transcriptional regulatory region obtained from an Arabidopsis oil body protein gene fused to (b) a DNA
sequence of interest heterologous to said regulatory region.
10. An expression cassette comprising:
as operably linked components, a transcriptional regulatory region obtained from an Arabidopsis oil body protein gene, a DNA sequence of interest heterologous to said regulatory region, and a transcriptional termination region.
as operably linked components, a transcriptional regulatory region obtained from an Arabidopsis oil body protein gene, a DNA sequence of interest heterologous to said regulatory region, and a transcriptional termination region.
11. A plant cell containing integrated into its genome a chimeric gene comprising (a) a transcriptional regulatory region obtained from an Arabidopsis oil body protein gene fused to (b) a DNA sequence of interest heterologous to said regulatory region.
12. A seed cell containing integrated into its genome a chimeric gene comprising (a) a transcriptional regulatory region obtained from an Arabidopsis oil body protein gene fused to (b) a DNA sequence of interest heterologous to said regulatory region.
13. A seed cell according to claim 12 wherein said cell is from a dicotyledonous seed.
14. A seed cell according to claim 12 wherein said cell is from an oilseed.
15. A method for altering seed-specific metabolism, said method comprising:
(a) transforming a plant capable of developing seed with an expression cassette comprising in the 5'-3' direction of transcription, a transcriptional initiation region obtained from an oil body protein gene, a DNA sequence of interest other than a sequence native to said initiation region wherein said DNA sequence of interest does not encode a plant acyl-ACP thioesterase or .beta.-ketoacyl-ACP synthetase II, and a transcriptional termination region; and (b) growing said plant under conditions whereby seed is produced in which said DNA sequence is expressed under transcriptional control of said transcriptional initiation region wherein expression of said DNA sequence alters seed-specific metabolism.
(a) transforming a plant capable of developing seed with an expression cassette comprising in the 5'-3' direction of transcription, a transcriptional initiation region obtained from an oil body protein gene, a DNA sequence of interest other than a sequence native to said initiation region wherein said DNA sequence of interest does not encode a plant acyl-ACP thioesterase or .beta.-ketoacyl-ACP synthetase II, and a transcriptional termination region; and (b) growing said plant under conditions whereby seed is produced in which said DNA sequence is expressed under transcriptional control of said transcriptional initiation region wherein expression of said DNA sequence alters seed-specific metabolism.
16. A method for altering seed-specific metabolism, said method comprising:
(a) transforming a plant capable of developing seed with an expression cassette comprising in the 5'-3' direction of transcription, a transcriptional initiation region obtained from an Arabidopsis oil body protein gene, a DNA sequence of interest other than a sequence native to said initiation region, and a transcriptional termination region; and (b) growing said plant under conditions whereby seed is produced in which said DNA sequence is expressed under transcriptional control of said transcriptional initiation region wherein expression of said DNA sequence alters seed-specific metabolism.
(a) transforming a plant capable of developing seed with an expression cassette comprising in the 5'-3' direction of transcription, a transcriptional initiation region obtained from an Arabidopsis oil body protein gene, a DNA sequence of interest other than a sequence native to said initiation region, and a transcriptional termination region; and (b) growing said plant under conditions whereby seed is produced in which said DNA sequence is expressed under transcriptional control of said transcriptional initiation region wherein expression of said DNA sequence alters seed-specific metabolism.
17. The method according to claim 15 or 16, wherein said altering is reducing or suppressing expression of endogenous genes expressed in plant seeds.
18. The method according to claim 15 or 16, wherein said transcriptional initiation region includes a silencer element.
19. The method according to claim 15 or 16, wherein a transcribed strand of said DNA sequence is complementary to mRNA endogenous to said cells.
20. A method for producing a heterologous polypeptide in seed, said method comprising:
(a) transforming a plant capable of developing seed with an expression cassette comprising in the 5'-3' direction of transcription, a transcriptional initiation region obtained from an oil body protein gene, a DNA sequence of interest encoding a polypeptide heterologous to said plant wherein said DNA sequence of interest does not encode a plant acyl-ACP thioesterase or .beta.-ketoacyl-ACP synthetase II, and a transcriptional termination region; and (b) growing said plant under conditions whereby seed is produced in which said DNA sequence is expressed under transcriptional control of said transcriptional initiation region.
(a) transforming a plant capable of developing seed with an expression cassette comprising in the 5'-3' direction of transcription, a transcriptional initiation region obtained from an oil body protein gene, a DNA sequence of interest encoding a polypeptide heterologous to said plant wherein said DNA sequence of interest does not encode a plant acyl-ACP thioesterase or .beta.-ketoacyl-ACP synthetase II, and a transcriptional termination region; and (b) growing said plant under conditions whereby seed is produced in which said DNA sequence is expressed under transcriptional control of said transcriptional initiation region.
21. A method for producing a heterologous polypeptide in seed, said method comprising:
(a) transforming a plant capable of developing seed with an expression cassette comprising in the 5'-3' direction of transcription, a transcriptional initiation region obtained from an Arabidopsis oil body protein gene, a DNA sequence of interest encoding a polypeptide heterologous to said plant, and a transcriptional termination region; and (b) growing said plant under conditions whereby seed is produced in which said DNA sequence is expressed under transcriptional control of said transcriptional initiation region.
(a) transforming a plant capable of developing seed with an expression cassette comprising in the 5'-3' direction of transcription, a transcriptional initiation region obtained from an Arabidopsis oil body protein gene, a DNA sequence of interest encoding a polypeptide heterologous to said plant, and a transcriptional termination region; and (b) growing said plant under conditions whereby seed is produced in which said DNA sequence is expressed under transcriptional control of said transcriptional initiation region.
22. A method for expressing a DNA sequence of interest in a host plant during a phase of embryogenesis which precedes accumulation of storage proteins, said method comprising:
transforming said host plant with a construct comprising a DNA
sequence of interest operably fused to a transcriptional regulatory region, wherein said regulatory region is obtained from an oil body protein gene and wherein said DNA sequence of interest does not encode a plant acyl-ACP thioesterase or .beta.-ketoacyl-ACP synthetase II; and growing said plant under conditions whereby seed is produced and said DNA sequence of interest in expressed under transcriptional control of said regulatory region.
transforming said host plant with a construct comprising a DNA
sequence of interest operably fused to a transcriptional regulatory region, wherein said regulatory region is obtained from an oil body protein gene and wherein said DNA sequence of interest does not encode a plant acyl-ACP thioesterase or .beta.-ketoacyl-ACP synthetase II; and growing said plant under conditions whereby seed is produced and said DNA sequence of interest in expressed under transcriptional control of said regulatory region.
23. The method according to claim 22 wherein said host is a dicotyledenous oil seed cell.
24. The method according to claim 18, wherein said oil body protein gene is obtained from a plant selected from the group consisting of (a) Brassica napus;
(b) Zea mays;
(c) carrot; and (d) Arabidopsis.
(b) Zea mays;
(c) carrot; and (d) Arabidopsis.
25. A method according to any one of claims 15-24 wherein said transcriptional regulatory region is obtained from an Arabidopsis oil body protein gene.
26. A method according to claim 2, 16, 21 or 25 wherein said Arabidopsis transcriptional regulatory region comprises nucleotides -867 to 1 of the sequence shown in Figure 2A.
27. A method according to claim 1 or 2 wherein said DNA sequence of interest encodes a fusion protein comprising an oil body protein.
28. A seed cell containing integrated into its genome a chimeric gene comprising (a) a transcriptional regulatory region obtained from an Arabidopsis oil body protein gene and (b) a DNA sequence of interest heterologous to said regulatory region wherein said heterologous DNA
sequence does not encode a plant acyl-ACP thioesterase or .beta.-ketoacyl-ACP synthetase II.
sequence does not encode a plant acyl-ACP thioesterase or .beta.-ketoacyl-ACP synthetase II.
29. A seed cell according to claim 28 wherein said cell is from a dicotyledonous seed.
30. A seed cell according to claim 28 wherein said cell is from an oilseed.
31. A DNA construct comprising:
a chimeric gene comprising (a) a transcriptional regulatory region obtained from an Arabidopsis oil body protein gene fused to (b) a DNA
sequence of interest heterologous to said regulatory region wherein said heterologous DNA sequence does not encode a plant acyl-ACP
thioesterase or .beta.-ketoacyl-ACP synthetase II.
a chimeric gene comprising (a) a transcriptional regulatory region obtained from an Arabidopsis oil body protein gene fused to (b) a DNA
sequence of interest heterologous to said regulatory region wherein said heterologous DNA sequence does not encode a plant acyl-ACP
thioesterase or .beta.-ketoacyl-ACP synthetase II.
32. An expression cassette comprising:
as operably linked components, a transcriptional regulatory region obtained from an Arabidopsis oil body protein gene, a DNA sequence of interest heterologous to said regulatory region wherein said heterologous DNA sequence does not encode a plant acyl-ACP thioesterase or .beta.-ketoacyl-ACP synthetase II, and a transcriptional termination region.
as operably linked components, a transcriptional regulatory region obtained from an Arabidopsis oil body protein gene, a DNA sequence of interest heterologous to said regulatory region wherein said heterologous DNA sequence does not encode a plant acyl-ACP thioesterase or .beta.-ketoacyl-ACP synthetase II, and a transcriptional termination region.
33. A plant cell containing integrated into its genome a chimeric gene comprising (a) a transcriptional regulatory region obtained from an oil body protein gene fused to (b) a DNA sequence of interest heterologous to said regulatory region wherein said heterologous DNA sequence does not encode a plant acyl-ACP thioesterase or .beta.-ketoacyl-ACP synthetase II.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US86235592A | 1992-04-02 | 1992-04-02 | |
| US07/862,355 | 1992-04-02 | ||
| PCT/CA1993/000141 WO1993020216A1 (en) | 1991-02-22 | 1993-04-02 | Oil-body protein cis-elements as regulatory signals |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2133417A1 CA2133417A1 (en) | 1993-10-14 |
| CA2133417C true CA2133417C (en) | 2005-12-06 |
Family
ID=35519624
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002133417A Expired - Lifetime CA2133417C (en) | 1992-04-02 | 1993-04-02 | Oil-body protein cis-elements as regulatory signals |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2133417C (en) |
-
1993
- 1993-04-02 CA CA002133417A patent/CA2133417C/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| CA2133417A1 (en) | 1993-10-14 |
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