CA2286515A1 - Constitutive expression of plant genes by a 648 bp regulatory sequence from a pea polyubiquitin gene - Google Patents
Constitutive expression of plant genes by a 648 bp regulatory sequence from a pea polyubiquitin gene Download PDFInfo
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- CA2286515A1 CA2286515A1 CA 2286515 CA2286515A CA2286515A1 CA 2286515 A1 CA2286515 A1 CA 2286515A1 CA 2286515 CA2286515 CA 2286515 CA 2286515 A CA2286515 A CA 2286515A CA 2286515 A1 CA2286515 A1 CA 2286515A1
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- promoter
- gene
- 35s35samv
- expression
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
Abstract
The invention disclosed is an Isolated DNA fragment for expressing gene function in plants, having a length of about 648 base pairs and being located in the PuBl polyubiquitin gene between the Hindlll and Xbal sites.
Description
CONSTITUTIVE EXPRESSION OF PLANT GENES BY A 648 by REGULATORY SEQUENCE FROM A
PEA POLYUBIQUITIN GENE.
BACKGROUND OF THE INVENTION
While structural genes contain the genetic information required for the production of functional proteins, regulatory sequences (promoters) that are located at the 5' end of the coding region play major roles in determining where, when, how strongly and in response to which chemical or environmental signals the gene will be expressed. Thus for transgenic modification of crop plants (genetic engineering) the genes must be matched with the appropriate regulatory sequences to achieve the desired control of gene function. Promoters have been defined as constitutive (gene expressed in all locations), tissue specific or conditional (responding to signals). However, even 'constitutive' regulatory sequences, may contain multiple independent elements, each controlling a different aspect of gene expression.
DESCRIPTION OF THE PRIOR ART
Although many upstream (5') regulatory sequences have been cloned and characterized with respect to their functional control of gene expression in plants, much transgenic experimentation has been based on the regulatory properties of a few patented 'constitutive' promoters including T-DNA promoters from Agrobacterium tumefaciens genes [US 4,771,002; US 5,102,796; US 5,504,200; US
5,591,605] such as nopaline synthase (NOS promoter) and octopine synthase (OCS promoter).
Constitutive promoters have also been found in plant viral sources and a strong constitutive promoter that controls the production of the 35S coat protein of Cauliflower Mosaic Virus has been used extensively to control the expression of plant genes [US 5,164,316; US 5,196,525; US 5,322,938; US 5,424,412; US 5,539,874; US
5,362,865; US 5,106,739]. In general, the CaMV35S promoter expresses genes at a greater level than the NOS
or OCS promoters. However, a tandem duplication of the CaMV35S sequence (Kay et a1.1987)2 increased gene expression ten-fold and the further addition of an Alfalfa Mosaic Virus (AMV) RNA4 untranslated leader sequence (Jobling and Gehrke)3 to the tandem CaMV35S promoter has increased gene expression to 30 times that produced by the CaMV35S
promoter. Nevertheless, the CaMV35S promoter itself has been used successfully to control the expression of plant genes in a wide range of transgenic applications and any new plant promoters should produce gene expression that is at least equivalent to this standard. Because of the patent protection associated with the above promoter systems, in 1994 we initiated a project at NRC's Plant Biotechnology Institute aimed at the identification of a novel constitutive promoter that would control plant gene expression at a level at least equivalent to that of the CaMV35S promoter. Dr. James Xia was appointed as a Research Associate to undertake this research effort. In parallel studies, another group of researchers identified an upstream untranscribed region of a maize ubiquitin gene and in 1996 published a report on their development of a set of ubiquitin promoter-based vectors for use in monocotyledonous plants. In the same year, they obtained a patent for a Plant ubiquitin promoter system(US 5,510,474). Since that time, they have extended their IP
protection in a series of patents.
SUMMARY OF THE INVENTION
Ubiquitin is a protein that is identical in sequence in all eukaryotic organisms°. In plants, ubiquitin genes are usually found as mufti-gene families. The individual genes contain a limited number of ubiquitin units, each with the identical 76 amino acids. The ubiquitin fusion polypeptides contain either multiple ubiquitins, linked head to tail (polyubiquitin) or ubiquitin monomers fused to one of two unrelated sequences, 52 or 81 amino acids in length. Although the ubiquitin protein is highly conserved, nucleotide sequences are quite divergent, especially in the 5' and 3' non-coding regions where gene regulatory sequences would be anticipated. Because of their ubiquitous distribution and common amino acid sequences, ubiquitin genes were selected as potential sources of constitutive promoter elements that could be used for controlling gene expression in transgenic plants.
To identify such regulatory regions, 4 polyubiquitin genes were isolated from an Alaska pea genomic library in ~. EMBL-3(Clontech, Palo Alto, CA; Cat. no. FL1101D) using a ubiquitin probe provided by Dr. Peter Quails.
The 4 genes (PUB1, PUB2, PUB3, PUB4) were completely sequenced and analyzed for functional elements6.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. General features of PUB 1 polyubiquitin gene, including the HindIII-XbaI region of the PUB 136HX
promoter, the intron, the 6 ubiquitin coding units and relevant restriction sites.
Figure 2. Sequence elements of PUB 136HX promoter DETAILED DESCRIPTION OF THE INVENTION
Examination of the 5' sequences of these genes identified possible regulatory elements, including a 29 by AT-rich sequence, two putative heat shock elements, an element with similarity to a G-box found in light-inducible plant tissues, and an exon/intron splice site (Figure 2). Therefore several sequences from the 5' regions of the 4 genes were fused to the uidA (gus) reporter gene8, in order to examine the relative effectiveness of these putative promoter regions, in relation to the well characterized strong, constitutive 35S35SAMV promoter2~3.
The levels of gene expression produced by these different constructs varied greatly, but of the constructs tested, the PUB 136HX promoter from pea polyubiquitin gene 1 (PUB 1 ) produced the most consistent and highest level of GUS expression in the assay systems that were used to compare the control of gene expression by the PUB136HX and 35S35SAMV promoters.
The PUB136HX promoter consists of a 648 by DNA sequence between the HindIII
and Xbal restriction sites (Figures 1 and 2). The promoter can be readily inserted into standard cloning vectors or 5' to the translational start site (ATG) of a gene. Thus the promoter consists of a 5' regulatory sequence, followed by 348 by of the intron, that in the native PUB 1 gene would terminate at the beginning of the first ubiquitin coding unit. Fusion of the PUB 136HX promoter to the uidA (gus) gene produced a reporter system that allows both quantitative fluorometric assays of GUS expression as well as colorimetric detection of GUS
expression in plant tissues.
The fusion of the PUB 136HX promoter to the uidA gene was used in the following experiments to compare its regulatory characteristics with those of the strong 35S35SAMV promoter.
Particle Bombardment Bacterial plasmids containing the GUS gene controlled by the PUB136HX and 35S35SAMV promoters, were coated onto gold particles and introduced into pea embryo axes and leaf discs using high pressure helium to accelerate the particles (Model PDS-1000/He Particle Delivery System, Bio-Rad Laboratories, 1000 Alfred Nobel Dr., Hercules, CA, USA). Twenty-four hours after shooting, the tissues were assayed for transient GUS
activity in the tissues using the quantitative fluorescence detection method of 4-methyl umbelliferone to estimate the ~i-glucuronidase activity in the tissues.' Significant GUS
activity was detected in both embryo axes and leaf discs of pea and with both promoters, although expression was greater with the 35S35SAMV promoter in both cases.
Leaves from Transgenic Peas Greenfeast peas were transformed using an Agrobacterium tumefaciens methods with the same promoter constructs controlling the GUS gene as used for particle bombardment. Two experiments were performed to assess the GUS expression in pea leaves, with Expt. 1 examining the original transgenic plant (To generation) and plants of the Ti generation. Expt. 2 re-examined the T, plants along with T2 plants. GUS activities in leaf tissues were significantly greater when controlled by the 35S35SAMV promoter than in plants transformed with the PUB 136HX promoter. The GUS activity with PUB 136HX was between 9 and 17 %
of the activity with the 35S35SAMV promoter.
Independent Transgenic Plants All of the plants examined in the "Leaves from Transgenic Peas" section above, were derived from the same original transgenic plant. However, several independent transgenic plants had been recovered from transformation with both the 35S35SAMV and PUB136HX promoters. GUS activities in leaves of 5 independent transgenic plants containing the uidA gene with one of the two gene promoters, verified the stronger GUS expression with the 35S35SAMV promoter. Over all of the experiments shown in Table 1, GUS
activity with PUB136HX averaged 13 +/-2 % of the stronger 35S35SAMV promoter.
All of the data in Table 1 were from peas, but it is also important to know if control of the GUS gene by the two promoters is expressed similarly with respect to different plant tissues.
Table 2. shows the GUS activity in nine plant tissues from transgenic peas with PUB136HX or 35S35SAMV controlling the uidA gene. In general, GUS
activities from the two promoters were strongly correlated (r = 0.93, P<0.001 ) with stamens exhibiting the highest levels of GUS expression and leaves having the least GUS expression with both promoters. Thus while expression from the PUB136HX promoter averaged only 30% of that from the 35S35SAMV promoter, the relative magnitude of GUS expression in different tissues was similar. Table 3 also shows greater GUS
expression from the 35S35SAMV promoter in transgenic tobacco and canola leaves, but in both species, GUS
activity from the PUB136HX promoter was greatly reduced compared to the 35S35SAMV promoter or the previous results with peas. As these results might indicate a relatively stronger gene expression in the legume, we used transient particle bombardment assays to compare the expression of the uidA gene with the two promoters in a larger group of crop species. The results (Table 4) showed differences between the two promoters, and among the species tested. GUS expression from the 35S35SAMV
promoter clearly differentiated between the the dicotyledonous and the monocotyledonous plants.
GUS activity in divots was three times that in monocots, whereas with the PUB 136HX promoter, there was no significance difference between the two taxonomic groups.
The results summarized in Tables 1 to 4, clearly indicate that the PUB 136HX
promoter produces significant GUS expression in all experimental systems, plant tissues and plant species that have been examined. Although the 35S35SAMV promoter produces consistently greater gene expression, the overall GUS expression by the PUB136HX promoter averaged 26 +/- 4 % of that produced by the strong 35S35SAMV
promoter in direct experimental comparisons. This level of expression is considerably superior to the CaMV35S promoter that has been widely used in plant biotechnology.
PEA POLYUBIQUITIN GENE.
BACKGROUND OF THE INVENTION
While structural genes contain the genetic information required for the production of functional proteins, regulatory sequences (promoters) that are located at the 5' end of the coding region play major roles in determining where, when, how strongly and in response to which chemical or environmental signals the gene will be expressed. Thus for transgenic modification of crop plants (genetic engineering) the genes must be matched with the appropriate regulatory sequences to achieve the desired control of gene function. Promoters have been defined as constitutive (gene expressed in all locations), tissue specific or conditional (responding to signals). However, even 'constitutive' regulatory sequences, may contain multiple independent elements, each controlling a different aspect of gene expression.
DESCRIPTION OF THE PRIOR ART
Although many upstream (5') regulatory sequences have been cloned and characterized with respect to their functional control of gene expression in plants, much transgenic experimentation has been based on the regulatory properties of a few patented 'constitutive' promoters including T-DNA promoters from Agrobacterium tumefaciens genes [US 4,771,002; US 5,102,796; US 5,504,200; US
5,591,605] such as nopaline synthase (NOS promoter) and octopine synthase (OCS promoter).
Constitutive promoters have also been found in plant viral sources and a strong constitutive promoter that controls the production of the 35S coat protein of Cauliflower Mosaic Virus has been used extensively to control the expression of plant genes [US 5,164,316; US 5,196,525; US 5,322,938; US 5,424,412; US 5,539,874; US
5,362,865; US 5,106,739]. In general, the CaMV35S promoter expresses genes at a greater level than the NOS
or OCS promoters. However, a tandem duplication of the CaMV35S sequence (Kay et a1.1987)2 increased gene expression ten-fold and the further addition of an Alfalfa Mosaic Virus (AMV) RNA4 untranslated leader sequence (Jobling and Gehrke)3 to the tandem CaMV35S promoter has increased gene expression to 30 times that produced by the CaMV35S
promoter. Nevertheless, the CaMV35S promoter itself has been used successfully to control the expression of plant genes in a wide range of transgenic applications and any new plant promoters should produce gene expression that is at least equivalent to this standard. Because of the patent protection associated with the above promoter systems, in 1994 we initiated a project at NRC's Plant Biotechnology Institute aimed at the identification of a novel constitutive promoter that would control plant gene expression at a level at least equivalent to that of the CaMV35S promoter. Dr. James Xia was appointed as a Research Associate to undertake this research effort. In parallel studies, another group of researchers identified an upstream untranscribed region of a maize ubiquitin gene and in 1996 published a report on their development of a set of ubiquitin promoter-based vectors for use in monocotyledonous plants. In the same year, they obtained a patent for a Plant ubiquitin promoter system(US 5,510,474). Since that time, they have extended their IP
protection in a series of patents.
SUMMARY OF THE INVENTION
Ubiquitin is a protein that is identical in sequence in all eukaryotic organisms°. In plants, ubiquitin genes are usually found as mufti-gene families. The individual genes contain a limited number of ubiquitin units, each with the identical 76 amino acids. The ubiquitin fusion polypeptides contain either multiple ubiquitins, linked head to tail (polyubiquitin) or ubiquitin monomers fused to one of two unrelated sequences, 52 or 81 amino acids in length. Although the ubiquitin protein is highly conserved, nucleotide sequences are quite divergent, especially in the 5' and 3' non-coding regions where gene regulatory sequences would be anticipated. Because of their ubiquitous distribution and common amino acid sequences, ubiquitin genes were selected as potential sources of constitutive promoter elements that could be used for controlling gene expression in transgenic plants.
To identify such regulatory regions, 4 polyubiquitin genes were isolated from an Alaska pea genomic library in ~. EMBL-3(Clontech, Palo Alto, CA; Cat. no. FL1101D) using a ubiquitin probe provided by Dr. Peter Quails.
The 4 genes (PUB1, PUB2, PUB3, PUB4) were completely sequenced and analyzed for functional elements6.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. General features of PUB 1 polyubiquitin gene, including the HindIII-XbaI region of the PUB 136HX
promoter, the intron, the 6 ubiquitin coding units and relevant restriction sites.
Figure 2. Sequence elements of PUB 136HX promoter DETAILED DESCRIPTION OF THE INVENTION
Examination of the 5' sequences of these genes identified possible regulatory elements, including a 29 by AT-rich sequence, two putative heat shock elements, an element with similarity to a G-box found in light-inducible plant tissues, and an exon/intron splice site (Figure 2). Therefore several sequences from the 5' regions of the 4 genes were fused to the uidA (gus) reporter gene8, in order to examine the relative effectiveness of these putative promoter regions, in relation to the well characterized strong, constitutive 35S35SAMV promoter2~3.
The levels of gene expression produced by these different constructs varied greatly, but of the constructs tested, the PUB 136HX promoter from pea polyubiquitin gene 1 (PUB 1 ) produced the most consistent and highest level of GUS expression in the assay systems that were used to compare the control of gene expression by the PUB136HX and 35S35SAMV promoters.
The PUB136HX promoter consists of a 648 by DNA sequence between the HindIII
and Xbal restriction sites (Figures 1 and 2). The promoter can be readily inserted into standard cloning vectors or 5' to the translational start site (ATG) of a gene. Thus the promoter consists of a 5' regulatory sequence, followed by 348 by of the intron, that in the native PUB 1 gene would terminate at the beginning of the first ubiquitin coding unit. Fusion of the PUB 136HX promoter to the uidA (gus) gene produced a reporter system that allows both quantitative fluorometric assays of GUS expression as well as colorimetric detection of GUS
expression in plant tissues.
The fusion of the PUB 136HX promoter to the uidA gene was used in the following experiments to compare its regulatory characteristics with those of the strong 35S35SAMV promoter.
Particle Bombardment Bacterial plasmids containing the GUS gene controlled by the PUB136HX and 35S35SAMV promoters, were coated onto gold particles and introduced into pea embryo axes and leaf discs using high pressure helium to accelerate the particles (Model PDS-1000/He Particle Delivery System, Bio-Rad Laboratories, 1000 Alfred Nobel Dr., Hercules, CA, USA). Twenty-four hours after shooting, the tissues were assayed for transient GUS
activity in the tissues using the quantitative fluorescence detection method of 4-methyl umbelliferone to estimate the ~i-glucuronidase activity in the tissues.' Significant GUS
activity was detected in both embryo axes and leaf discs of pea and with both promoters, although expression was greater with the 35S35SAMV promoter in both cases.
Leaves from Transgenic Peas Greenfeast peas were transformed using an Agrobacterium tumefaciens methods with the same promoter constructs controlling the GUS gene as used for particle bombardment. Two experiments were performed to assess the GUS expression in pea leaves, with Expt. 1 examining the original transgenic plant (To generation) and plants of the Ti generation. Expt. 2 re-examined the T, plants along with T2 plants. GUS activities in leaf tissues were significantly greater when controlled by the 35S35SAMV promoter than in plants transformed with the PUB 136HX promoter. The GUS activity with PUB 136HX was between 9 and 17 %
of the activity with the 35S35SAMV promoter.
Independent Transgenic Plants All of the plants examined in the "Leaves from Transgenic Peas" section above, were derived from the same original transgenic plant. However, several independent transgenic plants had been recovered from transformation with both the 35S35SAMV and PUB136HX promoters. GUS activities in leaves of 5 independent transgenic plants containing the uidA gene with one of the two gene promoters, verified the stronger GUS expression with the 35S35SAMV promoter. Over all of the experiments shown in Table 1, GUS
activity with PUB136HX averaged 13 +/-2 % of the stronger 35S35SAMV promoter.
All of the data in Table 1 were from peas, but it is also important to know if control of the GUS gene by the two promoters is expressed similarly with respect to different plant tissues.
Table 2. shows the GUS activity in nine plant tissues from transgenic peas with PUB136HX or 35S35SAMV controlling the uidA gene. In general, GUS
activities from the two promoters were strongly correlated (r = 0.93, P<0.001 ) with stamens exhibiting the highest levels of GUS expression and leaves having the least GUS expression with both promoters. Thus while expression from the PUB136HX promoter averaged only 30% of that from the 35S35SAMV promoter, the relative magnitude of GUS expression in different tissues was similar. Table 3 also shows greater GUS
expression from the 35S35SAMV promoter in transgenic tobacco and canola leaves, but in both species, GUS
activity from the PUB136HX promoter was greatly reduced compared to the 35S35SAMV promoter or the previous results with peas. As these results might indicate a relatively stronger gene expression in the legume, we used transient particle bombardment assays to compare the expression of the uidA gene with the two promoters in a larger group of crop species. The results (Table 4) showed differences between the two promoters, and among the species tested. GUS expression from the 35S35SAMV
promoter clearly differentiated between the the dicotyledonous and the monocotyledonous plants.
GUS activity in divots was three times that in monocots, whereas with the PUB 136HX promoter, there was no significance difference between the two taxonomic groups.
The results summarized in Tables 1 to 4, clearly indicate that the PUB 136HX
promoter produces significant GUS expression in all experimental systems, plant tissues and plant species that have been examined. Although the 35S35SAMV promoter produces consistently greater gene expression, the overall GUS expression by the PUB136HX promoter averaged 26 +/- 4 % of that produced by the strong 35S35SAMV
promoter in direct experimental comparisons. This level of expression is considerably superior to the CaMV35S promoter that has been widely used in plant biotechnology.
Table 1. Relative GUS Expression from PUB136HX and 35S35SAMV Promoters PUB136HX 35S35SAMV Percent of (pmol min-~ mg protein '~) 35S35SAMV
Bombardment Embryo Axes 64 302 21 Leaf Discs 4 74 5 Transgeruc Pea Leaves Expt 1 To 1245 9750 13 T, 2133 23281 9 Expt 2 T, 673 3908 17 Independent Insertions 1. 594 4550 2. 267 4529 3. 177 4487 4. 82 3589 5. 32 2123 Mean 538 +/- 196 5501 +/- 1939 10 Overall Mean 13 +/-Table Relative GUS Expression 2. in Pea Tissues from PUB136HX
and 35S35SAMV promoters, with tissues listed by decreasing order of GUS
expression with promoter TISSUE PUB136HX 35S35SAMV PUB136HX % of (nmol miri ~ m g protein '~) 35S35SAMV
Mean Mean Stamens12.94 39.87 33 Petals 3.51 17.19 20 Roots 3.30 16.84 20 Sepals 4.08 13.48 30 Stem 1.74 12.14 14 Pistil 3.35 11.52 29 Seed 2.41 5.66 67 Pod 2.87 4.26 43 Leaf 0.76 4.06 19 Mean 3.88 +/- 1.18 13.9 +/- 3.7 30 +/- 5 Table 3. GUS Activity from PUB136HX and 35S35SAMV Promoters in Leaf Discs From Transgenic Tobacco and Canola Plants PUB136HX 35S35SAMV PUB136HX % of (pmol MU.mici ~ mg protein -~) 35S35SAMV
Tobacco Expt 1. 117 15205 0.8 Expt 2. 34 11041 0.3 Canola 219 6637 3.3 Table 4. GUS Activity From PUB136HX and 35S35SAMV Promoters. Plasmids Carrying the 2 Constructs Were Introduced Into Leaf Discs From Nine Crop Species by Particle Bombardment.
PROMOTER 35S35SAMV PUB136HX PUB136HX % of .(pmol. MU miri ~. MgProteili ~) 35S35SAMV
Dicotyledons Pea 27.8 8.7 31 Lentil 18.5 6.8 36 Chickpea 8.9 6.0 67 Bean 62.8 16.3 26 Tobacco 24.6 11.1 45 Canola 17.7 8.2 46 Mean 26.8 +/- 7.7 9.6 +/- 1.5 42 +/- 6 Monocotyledons Wheat 8.2 7.0 85 Barley 6.3 8.2 130 Corn 7.3 8.8 121 Mean 7.3 +/- 0.6 8.0 +/- 0.5 112 +/- 13 Dicot vs Monocot P=0.02 NS P< .001 REFERENCES FOR PATENT DOCUMENT
'Benfey, P.N. and Nam-Hai Chua (1990). The cauliflower mosaic virus 35S
promoter:
combinatorial regulation of transcription in plants. Science 250:959-966 = Kay, Robert, Amy Chan, Mark Daly, Joan McPherson (1987). Duplication of CaMV
promoter sequences creates a strong enhancer for plant genes. Science 236:
' Jobling, Stephen A. and Lee Gehrke (1987). Enhanced translation of chimaeric messenger RNAs containing a plant viral untranslated leader sequence. Nature 325:622-625 ' Callis, J. and R.D. Vierstra (1989). Ubiquitin and ubiquitin genes in higher plants. Oxford Surv.
Plant Mol. Cell Biol. 6: 1-30 SChristiansen, A.H. and P.H.Quail (1989). Sequence analysis and transcriptional regulation by heat shock of polyubiquitin transcripts from maize. Plant Mol. Biol. 12: 619-Xia, X. and J. D. Mahon (1998). Pea polyubiquitin genes: (I) structure and genomic organization. Gene 215: 445-452 'Christiansen, A.H. and P.H. Quail (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants.
Transgenic Research 5:213-218.
°Jefferson, R.A. T.A. Kavanagh and M.W. Bevan (1987) GUS fusions: (3-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901-°Schroeder, H.E., A.H. Schotz, T. Wardley-Richardson, D. Spencer, T.J.V. Higgins (1993).
Transformation and regeneration of two cultivars of pea (Pisum Sativum L.) Plant Physiology 101: 751-757.
'°Giuliano, G., E. Pichersky, V.S. Malik, M.P. Timko, P.A. Scolnik, A.R. Cashmore (1998). An evolutionarily conserved protein binding sequence upstream of a plant light-regulated gene.
Proc. Natl. Acad. Sci. USA, 85:7089-7093.
b
Bombardment Embryo Axes 64 302 21 Leaf Discs 4 74 5 Transgeruc Pea Leaves Expt 1 To 1245 9750 13 T, 2133 23281 9 Expt 2 T, 673 3908 17 Independent Insertions 1. 594 4550 2. 267 4529 3. 177 4487 4. 82 3589 5. 32 2123 Mean 538 +/- 196 5501 +/- 1939 10 Overall Mean 13 +/-Table Relative GUS Expression 2. in Pea Tissues from PUB136HX
and 35S35SAMV promoters, with tissues listed by decreasing order of GUS
expression with promoter TISSUE PUB136HX 35S35SAMV PUB136HX % of (nmol miri ~ m g protein '~) 35S35SAMV
Mean Mean Stamens12.94 39.87 33 Petals 3.51 17.19 20 Roots 3.30 16.84 20 Sepals 4.08 13.48 30 Stem 1.74 12.14 14 Pistil 3.35 11.52 29 Seed 2.41 5.66 67 Pod 2.87 4.26 43 Leaf 0.76 4.06 19 Mean 3.88 +/- 1.18 13.9 +/- 3.7 30 +/- 5 Table 3. GUS Activity from PUB136HX and 35S35SAMV Promoters in Leaf Discs From Transgenic Tobacco and Canola Plants PUB136HX 35S35SAMV PUB136HX % of (pmol MU.mici ~ mg protein -~) 35S35SAMV
Tobacco Expt 1. 117 15205 0.8 Expt 2. 34 11041 0.3 Canola 219 6637 3.3 Table 4. GUS Activity From PUB136HX and 35S35SAMV Promoters. Plasmids Carrying the 2 Constructs Were Introduced Into Leaf Discs From Nine Crop Species by Particle Bombardment.
PROMOTER 35S35SAMV PUB136HX PUB136HX % of .(pmol. MU miri ~. MgProteili ~) 35S35SAMV
Dicotyledons Pea 27.8 8.7 31 Lentil 18.5 6.8 36 Chickpea 8.9 6.0 67 Bean 62.8 16.3 26 Tobacco 24.6 11.1 45 Canola 17.7 8.2 46 Mean 26.8 +/- 7.7 9.6 +/- 1.5 42 +/- 6 Monocotyledons Wheat 8.2 7.0 85 Barley 6.3 8.2 130 Corn 7.3 8.8 121 Mean 7.3 +/- 0.6 8.0 +/- 0.5 112 +/- 13 Dicot vs Monocot P=0.02 NS P< .001 REFERENCES FOR PATENT DOCUMENT
'Benfey, P.N. and Nam-Hai Chua (1990). The cauliflower mosaic virus 35S
promoter:
combinatorial regulation of transcription in plants. Science 250:959-966 = Kay, Robert, Amy Chan, Mark Daly, Joan McPherson (1987). Duplication of CaMV
promoter sequences creates a strong enhancer for plant genes. Science 236:
' Jobling, Stephen A. and Lee Gehrke (1987). Enhanced translation of chimaeric messenger RNAs containing a plant viral untranslated leader sequence. Nature 325:622-625 ' Callis, J. and R.D. Vierstra (1989). Ubiquitin and ubiquitin genes in higher plants. Oxford Surv.
Plant Mol. Cell Biol. 6: 1-30 SChristiansen, A.H. and P.H.Quail (1989). Sequence analysis and transcriptional regulation by heat shock of polyubiquitin transcripts from maize. Plant Mol. Biol. 12: 619-Xia, X. and J. D. Mahon (1998). Pea polyubiquitin genes: (I) structure and genomic organization. Gene 215: 445-452 'Christiansen, A.H. and P.H. Quail (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants.
Transgenic Research 5:213-218.
°Jefferson, R.A. T.A. Kavanagh and M.W. Bevan (1987) GUS fusions: (3-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901-°Schroeder, H.E., A.H. Schotz, T. Wardley-Richardson, D. Spencer, T.J.V. Higgins (1993).
Transformation and regeneration of two cultivars of pea (Pisum Sativum L.) Plant Physiology 101: 751-757.
'°Giuliano, G., E. Pichersky, V.S. Malik, M.P. Timko, P.A. Scolnik, A.R. Cashmore (1998). An evolutionarily conserved protein binding sequence upstream of a plant light-regulated gene.
Proc. Natl. Acad. Sci. USA, 85:7089-7093.
b
Claims (2)
1. An isolated DNA fragment for effecting gene expression in plants, having a length of about 648 base pairs and being located in the PUBI
polyubiquitin gene between the Hindlll and Xbal sites.
polyubiquitin gene between the Hindlll and Xbal sites.
2. A DNA fragment according to Claim 1, as illustrated in Figure 2.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2286515 CA2286515A1 (en) | 1999-10-27 | 1999-10-27 | Constitutive expression of plant genes by a 648 bp regulatory sequence from a pea polyubiquitin gene |
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| Application Number | Priority Date | Filing Date | Title |
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| CA 2286515 CA2286515A1 (en) | 1999-10-27 | 1999-10-27 | Constitutive expression of plant genes by a 648 bp regulatory sequence from a pea polyubiquitin gene |
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|---|---|
| CA2286515A1 true CA2286515A1 (en) | 2001-04-27 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2286515 Abandoned CA2286515A1 (en) | 1999-10-27 | 1999-10-27 | Constitutive expression of plant genes by a 648 bp regulatory sequence from a pea polyubiquitin gene |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2286515A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023084416A1 (en) * | 2021-11-09 | 2023-05-19 | Benson Hill, Inc. | Promoter elements for improved polynucleotide expression in plants |
-
1999
- 1999-10-27 CA CA 2286515 patent/CA2286515A1/en not_active Abandoned
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023084416A1 (en) * | 2021-11-09 | 2023-05-19 | Benson Hill, Inc. | Promoter elements for improved polynucleotide expression in plants |
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