CA2168617C - Constitutive promoter from tobacco - Google Patents

Abstract

T-DNA tagging with a promoterless .beta.-glucuronidase (GUS) gene generated a transgenic Nicotiana tabacum plant that expressed GUS activity constitutively. The gene fusion has been cloned and sequenced. It has been re-inserted into N. tabacum by Agrobacterium-mediated transformation. The N. tabacum DNA upstream from the GUS gene was approximately 2 kb in length and showed no homology to known sequences. This DNA, which contains a constitutive promoter, is useful in controlling the expression of exogenous genes in transgenic plants of diverse plant species.

Description

CONSTITUTIVE PROMOTER FROM TOBACCO
FIELD OF INVENTION
The present invention relates to a constitutive promoter identified from Nicotiana tabacum (tobacco). This invention further relates to the use of said constitutive promoter to control the expression of exogenous genes in transgenic plants of diverse plant species.
BACKGROUND OF THE INVENTION
Bacteria from the genus Agrobacterium have the ability to transfer specific segments of DNA (T-DNA) to plant cells, where they stably integrate into the nuclear chromosomes. Analyses of plants harbouring the T-DNA
have revealed that this genetic element may be integrated at numerous locations, and can occasionally be found within genes. One strategy which has been exploited to identify integration events within genes is to transform plant cells with specially designed T-DNA vectors which contain a reporter gene, devoid of cis-acting transcriptional and translational expression signals (i.e. promoterless), located at the end of the T-DNA. Upon integration, the initiation codon of the promoterless gene (reporter gene) will be juxtaposed to plant sequences. The consequence of T-DNA insertion adjacent to, and downstream of, gene promoter elements may be the activation of reporter gene expression. The resulting hybrid genes, referred to as T-DNA-mediated gene fusions, consist of unknown and thus un-characterized plant promoters residing at their natural location within the chromosome, and the coding sequence of a marker gene located on the inserted T-DNA (Fobert et al. , 1991, Plant Mol. Biol. 17, 837-851).
It has generally been assumed that activation of promoterless or enhancerless marker genes result from T-DNA insertions within or immediately adjacent to genes. The recent isolation of several T-DNA
insertional mutants (Koncz et al., 1992, Plant Mol. Biol. 20, 963-976;

reviewed in Feldmann, 1991, Plant J. 1, 71-82; Van Lijsebettens et al., 1991, Plant Sci. 80, 27-37; Walden et al., 1991, Plant J. 1: 281-288; Yanofsky et al., 1990, Nature 346, 35-39), shows that this is the case for at least some insertions. However, other possibilities exist. One of these possibilities is that integration of the T-DNA activates silent regulatory sequences that are not associated with genes. Lindsey et al. (1993, Transgenic Res. 2, 33-47) referred to such sequences as "pseudo-promoters" and suggested that they may be responsible for activating marker genes in some transgenic lines. Fobert et al. (1994, Plant J. 6, 567-577) have cloned such sequences and have referred to these as "cryptic promoters" .
SUMMARY OF THE INVENTION
The present invention is directed to a constitutive promoter identified from Nicotiana tabacum (tobacco).
The transgenic tobacco plant, T1275, contained a 4.2 kb EcoRIlXbaI fragment containing the 2.2 kb promoterless GUS-nos gene and 2.0 kb of 5' flanking tobacco DNA. This 5' flanking DNA showed no homology to known sequences. Expression of the cloned fragment in transgenic tobacco is apparent on cultured leaf discs and in the early stages of shoot development.
Thus according to the present invention there is provided a constitutive promoter from tobacco. The present invention is further directed to a constitutive promoter having a DNA sequence, substantially homologous to SEQ ID NO: 1.
This invention also relates to a chimeric gene construct comprising:
a constitutive promoter, having a DNA sequence substantially homologous to SEQ ID NO: 1, and a gene encoding a protein, for which constitutive expression is desired.

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This invention further relates to a cloning vector containing said chimeric gene construct.
This invention also includes a plant cell which has been transformed with said cloning vector.
This invention further relates to a transgenic plant containing a constitutive promoter, having a DNA sequence substantially homologous to SEQ ID NO: 1, operatively linked to a gene encoding a protein.
Also included in the present invention is a method of conferring constitutive expression on a gene in a plant, comprising: operatively linking an exogenous gene, for which constitutive expression is desired, with a constitutive promoter, to produce a chimeric gene construct and introducing the chimeric gene construct into a plant capable of expressing the chimeric gene construct.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
FIGURE 1 shows the constitutive expression of GUS in all tissues of plant T1275, including leaf segments (a), stem cross-sections (b), roots (c), flower cross-sections (d), ovary cross-sections (e), immature embryos (f), mature embryos (g), and seed cross-sections (h).
FIGURE 2 shows the GUS fluorogenic activity, which reveals that the level of GUS expression in T1275 is comparable to levels in plants expressing CaMV 35S - GUS - nos genes in leaf tissues.

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FIGURE 3 is the Southern blot analysis of Eco RI digested T1275 DNA with a GUS gene coding region probe (lane 1) and a nptll gene coding region probe (lane 2).
FIGURE 4 shows the cloned GUS gene fusion pT1275.
FIGURE 5 shows the nucleotide sequence for the Xba I - Sal I
fragment of pT1275.
DESCRIPTION OF PREFERRED EMBODIMENT
The present invention relates to plant gene promoters. Specifically this invention relates to a constitutive promoter, identified by T-DNA tagging with a promoterless ~i-glucuronidase gene (GUS) to generate a transgenic N.
tabacum plant that expresses GUS activity constitutively.
In the context of this disclosure, the term "promoter" or "promoter region" refers to a sequence of DNA, usually upstream (5') to the coding sequence of a structural gene, which controls the expression of the coding region by providing the recognition for RNA polymerase and/or other factors required for transcription to start at the correct site.
There are generally two types of promoters, inducible and constitutive promoters. An inducible promoter is a promoter that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer the DNA
sequences or genes will not be transcribed. Typically the protein factor, that binds specifically to an inducible promoter to activate transcription, is present in an inactive form which is then directly or indirectly converted to the active form by the inducer. The inducer can be a chemical agent such as a protein, metabolite, growth regulator, herbicide or phenolic compound or a physiological stress imposed directly by heat, cold, salt, or toxic elements or ~16~fi1 indirectly through the action of a pathogen or disease agent such as a virus.
A plant cell containing an inducible promoter may be exposed to an inducer by externally applying the inducer to the cell or plant such as by spraying, watering, heating or similar methods.
The present invention is directed to a constitutive promoter which directs the expression of a gene, constitutively. Specifically, the present invention is directed to a constitutive promoter isolated from N. tabacum. A
constitutive promoter directs the expression of a gene throughout the various parts of a plant and continuously throughout plant development. Examples of known constitutive promoters include those associated with the CaMV 35S
transcript and Agrobacterium Ti plasmid nopaline synthase gene (Sanders et al. 1987, Nucleic Acids Res. 15, 1543-1558). The constitutive promoter of the present invention demonstrated levels of expression greater than that using the CaMV 35S promoter.
The term "constitutive" as used herein does not necessarily indicate that a gene is expressed at the same level in all cell types, but that the gene is expressed in a wide range of cell types, although some variation in abundance is often observed.
The present invention is further directed to a chimeric gene construct containing a gene of interest operatively linked to the constitutive promoter of the present invention. Any exogenous gene can be used and manipulated according to the present invention to result in the constitutive expression of said exogenous gene.
The chimeric gene construct of the present invention can further comprise a 3' untranslated region. A 3' untranslated region refers to that portion of a gene comprising a DNA segment that contains a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by ~16~61'~

effecting the addition of polyadenylic acid tracks to the 3' end of the mRNA
precursor. Polyadenylation signals are commonly recognized by the presence of homology to the canonical form 5' AATAAA-3' although variations are not uncommon.
Examples of suitable 3' regions are the 3' transcribed non-translated regions containing a polyadenylation signal of Agrobacterium tumor inducing (Ti) plasmid genes, such as the nopaline synthase (Nos gene) and plant genes such as the soybean storage protein genes and the small subunit of the ribulose-1, 5-bisphosphate carboxylase (ssRUBISCO) gene. The 3' untranslated region from the structural gene of the present construct can therefore be used to construct chimeric genes for expression in plants.
The chimeric gene construct of the present invention can also include further enhancers, either translation or transcription enhancers, as may be required. These enhancer regions are well known to persons skilled in the art, and can include the ATG initiation codon and adjacent sequences. The initiation codon must be in phase with the reading frame of the coding sequence to ensure translation of the entire sequence. The translation control signals and initiation codons can be from a variety of origins, both natural and synthetic. Translational initiation regions may be provided from the source of the transcriptional initiation region, or from the structural gene. The sequence can also be derived from the promoter selected to express the gene, and can be specifically modified so as to increase translation of the mRNA.
To aid in identification of transformed plant cells, the constructs of this invention may be further manipulated to include plant selectable markers.
Useful selectable markers include enzymes which provide for resistance to an antibiotic such as gentamycin, hygromycin, kanamycin, and the like.
Similarly, enzymes providing for production of a compound identifiable by colour change such as GUS (/3-glucuronidase), or luminescence, such as luciferase are useful.

Also considered part of this invention are transgenic plants containing the chimeric gene construct of the present invention. Methods of regenerating whole plants from plant cells are known in the art, and the method of obtaining transformed and regenerated plants is not critical to this invention. In general, transformed plant cells are cultured in an appropriate medium, which may contain selective agents such as antibiotics, where selectable markers are used to facilitate identification of transformed plant cells. Once callus forms, shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and the shoots transferred to rooting medium for regeneration of plants. The plants may then be used to establish repetitive generations, either from seeds or using vegetative propagation techniques.
The constructs of the present invention can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA
transformation, micro-injection, electroporation, etc. For reviews of such techniques see for example Weissbach and Weissbach, Methods for Plant Molecular Biology, Academy Press, New York VIII, pp. 421-463 (1988); and Geierson and Corey, Plant Molecular Biology, 2d Ed. (1988). The present invention further includes a suitable vector comprising the chimeric gene construct.
When specific sequences are referred to in the present invention, it is understood that these sequences include within their scope sequences that are "substantially homologous" to said specific sequences. Sequences are "substantially homologous" when at least about 80%, preferably at least about 90 % and most preferably at least about 95 % of the nucleotides match over a defined length of the molecule. Sequences that are "substantially homologous" include any substitution, deletion, or addition within the sequence. DNA sequences that are substantially homologous can be identified in Southern hybridization experiments, for example under stringent _8_ hybridization conditions (see Maniatis et al., in Molecular Cloning (A
Laboratory Manual), Cold Spring Harbor Laboratory (1982) p 387 to 389). For example, hybridization is done in 6 x SSC, O.O1M EDTA, 5 x Denhardt's solution, 0.5% SDS and 100~g/ml denatured salmon sperm DNA, in addition to the DNA probe, at 68°C for 3-16 hours, as needed. The filter is S then washed for 5 minutes in 2 x SSC and 0.5% SDS at room temperature, followed by a second wash at room temperature in 0.1 x SSC and 0.1 % SDS for 1 S minutes. A
final wash is done in 0.1 x SSC and 0.5% SDS at 68°C for 2 hours.
The specific sequences, referred to in the present invention, also include sequences which are "functionally equivalent" to said specific sequences. In the present invention functionally equivalent sequences refer to sequences which although not identical to the specific sequences provide the same or substantially the same function. DNA
sequences that are functionally equivalent include any substitution, deletion or addition within the sequence.
With reference to the present invention functionally equivalent sequences will direct the expression of an exogenous gene constitutively.
While this invention is described in detail with particular reference to preferred embodiments thereof, said embodiments are offered to illustrate but not limit the invention.
EXAMPLES
Characterization of a Constitutive promoter - GUS Fusion Transfer of binary constructs to Agrobacterium and leaf disc transformation of N.
tabacum SR1 were performed as described by Fobert et al. (1991, Plant Mol.
Biol. 17, 837-851). Plant tissue was maintained on 100 p,g/ml kanamycin sulfate (Sigma) throughout in vitro culture.
From the transgenic plants produced, one of these, T1275, was chosen for detailed study because of its high level and constitutive expression of GUS.
Fluorogenic and histological GUS assays were performed according to Jefferson (Plant Mol. Biol. Rep., 1987, 5, 387-405), as modified by Fobert et al. (Plant Mol. Biol., 1991, 17, 837-851). For initial screening, leaves were harvested from in vitro grown plantlets. Later nine different tissues:

216~61'~

leaf (L), stem (S), root (R), anther (A), petal (P), ovary (O), sepal (Se), seeds days post anthesis (S1) and seeds 20 days post-anthesis (S2), were collected from plants grown in the greenhouse and analyzed. For detailed, quantitative analysis of GUS activity, leaf, stem and root tissues were collected from 5 kanamycin resistant F1 progeny grown in vitro. Floral tissues were harvested at developmental stages 8-10 (Koltunow et al., 1990, Plant Cell 2, 1201-1224) from the original transgenic plants. Flowers were also tagged and developing seeds were collected from capsules at 10 and 20 dpa. In all cases, tissue was weighed, immediately frozen in liquid nitrogen, and stored at -80°C.
Tissues analyzed by histological assay were at the same developmental stages as those listed above. Different hand-cut sections were analyzed for each organ. For each plant, histological assays were performed on at least two different occasions to ensure reproducibility. Except for floral organs, all tissues were assayed in phosphate buffer according to Jefferson (1987, Plant Mol. Biol. Rep. 5, 387-405), with 1 mM X-Gluc (Sigma) as substrate. Flowers were assayed in the same buffer containing 20% (v/v) methanol (Kosugi et al., 1990, Plant Sci. 70, 133-140).
GUS activity in plant T1275 was found in all tissues. Figure 1 shows the constitutive expression of GUS by histochemical staining with X-Gluc of T1275, including leaf (a), stem (b), root (c), flower (d), ovary (e), embryos (f and g), and seed (h).
Constitutive GUS expression was confirmed with the more sensitive fluorogenic assay of plant tissue from transformed plant T1275. These results are shown in Figure 2. GUS expression was evident in all tissue types including leaf (L), stem (S), root (R), anther (A), pistil (P), ovary (O), sepal (Se), seeds at 10 dpa (S1) and 20 dpa (S2). Furthermore, the level of GUS
expression is comparable to the level of expression in transformed plants containing the constitutive promoter CaMV 35S in a GUS - nos fusion. As reported by Fobert et al. (1991, Plant Molecular Biology, 17: 837-851) GUS

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-lo-activity in transformed plants containing pBI121 (Clontech), which contains a CaMV 35S - GUS - nos chimeric gene, was as high as 18,770 ~ 2450 (pmole MU per minute per mg protein).
Genetic Analysis of Transgenic Plant T1275 The T-DNA contains a kanamycin resistance gene. Seeds from self pollinated transgenic plants were surface-sterilized in 70% ethanol for 1 min and in undiluted Javex bleach (6% sodium hypochloride) for 25 min.
Seeds were then washed several times with sterile distilled water, dried under laminar flow, and placed in Petri dishes containing MSO medium supplemented with 100 ~.g/ml kanamycin as described in Miki et al. (1993, Methods in Plant Molecular Biology and Biotechnology, Eds., B.R. Glick and J.E. Tompson, CRC Press, Boca Raton, 67-88). At least 90 plantlets were counted for each transformant. The number of green (kanamycin-resistant) and bleached (kanamycin-sensitive) plantlets were counted after 4-6 weeks, and analyzed using the Chit test at a significance level of P<0.05.
The genetic analysis results are shown below in Table 1, which demonstrates that the T-DNA loci segregated as a single locus of insertion.

Genetic Analysis of Transgenic Plant T1275 No. of No. of Observed Expected Chit Progeny Progeny Ratio Ratio Km' Km' 262 88 3:1' 3:1 0 ' Consistent with a single dominant gene Southern Blot Analysis The T-DNA in the transgenic plant T1275 was analyzed using either a GUS gene coding region probe or a nptll gene coding region probe.
Genomic DNA was isolated from freeze-dried leaves using the protocol of Sanders et al. (1987, Nucleic Acid Res. 15, 1543-1558). Ten micrograms of T1275 DNA was digested for several hours with EcoRI using the appropriate manufacturer-supplied buffer supplemented with 2.5 mM
spermidine. After electrophoresis through a 0. 8 % TAE agarose gel, Southern blot analysis was conducted using standard protocols. As the T-DNA from the construct containing the constitutive promoter - GUS - nos construct contains only a single Eco RI recognition site the hybridizing fragments are composed of both T-DNA and flanking tobacco DNA sequences. The length of the fragment will vary depending on the location of the nearest Eco RI
site.
~ Using the GUS gene as a probe (Figure 3 - lane 1), the fragment to the nearest Eco RI site in the plant DNA will be detected. With T1275, one such fragment was located. Using the nptll coding region as a probe (Figure 3 -lane 2), which hybridizes to sequences on the opposite side of the Eco RI
site, again only one hybridization band was evident. As can also be seen in Figure 3, no major rearrangements occurred.
Cloning and Analysis of the Constitutive Promoter - GUS Vision Genomic DNA was isolated from leaves according to Hattori et al.
(1987, Anal. Biochem. 165, 70-74). Ten ~cg of T1275 total DNA was digested with EcoRI and XbaI according to the manufacturer's instructions.
The digested DNA was size-fractionated on a 0.7 % agarose gel. The DNA
fragments of about 4 to 6 kb were isolated from the gel using the Elu-Quick#
kit (Schleicher and Schuell) and ligated to lambdaGEM-2 arms previously digested with EcoRI and XbaI and phosphatase-treated. About 40,000 plaques were transferred to a nylon membrane (Hybond#Amersham) and screened with the 3zP-labelled 2kb GUS insert isolated form pBIl2l, essentially as Trademark described in Rutledge et al. (1991, Mol. Gen Genet. 229, 31-40). The positive clones were isolated. The XbaI-EcoRI fragment (Figure 4) was isolated from the lambda phage and cloned into pTZl9R previously digested with XbaI and EcoRI and treated with intestinal calf phosphatase.
The 4.2kb fragment containing about 2.2kb of the T1275 promoter activity fused to the GUS gene and the nos 3' was isolated by digesting pTZ-T1275 with HindIII and EcoRI. The isolated fragment was ligated into the pRD400 vector (Datla et al. , 1992, Gene, 211:383-384) previously digested with HindIII and EcoRI and treated with calf intestinal phosphatase. Transfer of the binary vector to Agrobacterium tumefacierrs and leaf disc transformation of N. tabacum SR1 were performed as described above. Histochemical analysis of GUS activity revealed staining of callus tissue after transformation and prior to shoot organogenesis and staining of hoots subsequently. Staining ~ was comparable for shoots transformed with a vector in which the T1275 promoter was replaced with the 3kb 35S promoter from pBI121.
The present invention has been described with regard to preferred embodiments. However, it will be obvious to persons skilled in the art that a number of .variations and modifications can be made without departing from the scope of the invention as described in the following claims.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: BRIAN MIKI
(B) STREET: 1876 Dorset Drive (C) CITY: Ottawa (D) STATE: Ontario (E) COUNTRY: Canada (F) POSTAL CODE (ZIP): K1H 5V1 (A) NAME: JIRO HATTORI
(B) STREET: 763 Halstead Street (C) CITY: Ottawa (D) STATE: Ontario (E) COUNTRY: Canada (F) POSTAL CODE (ZIP): K1G 1M5 (A) NAME: PIERRE FOBERT
(B) STREET: 878 Kingsmere Blvd.
(C) CITY: Saskatoon (D) STATE: Saskatchewan (E) COUNTRY: Canada (F) POSTAL CODE (ZIP): S7J 4J7 (A) NAME: VENKATRAN N. IYER
(B) STREET: 139 Iona Street (C) CITY: Ottawa (D) STATE: Ontario (E) COUNTRY: Canada (F) POSTAL CODE (ZIP): K1Y 3M2 (ii) TITLE OF INVENTION: Constitutive Promoter (iii) NUMBER OF SEQUENCES: 1 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2255 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

21~'t ~ 1'~

TCTTAGGCAGGAAAAATCTTCTATTGGGCCATAATAAGAACTACCAATTAGAAAGGAGGT 1200 '

Claims (12)

1. A promoter comprising the nucleotide sequence, or the compliment of the nucleotide sequence, defined in SEQ ID NO:1.

2. A promoter comprising a nucleotide sequence that is from 80% to 100%
identical to SEQ ID NO. 1, or its compliment, over the length of the nucleotide sequence, wherein the promoter is a constitutive promoter.

3. A promoter comprising a nucleotide sequence that is from 90% to 100%
identical to SEQ ID NO. 1, or its compliment, over the length of the nucleotide sequence, wherein the promoter is a constitutive promoter.

4. A promoter comprising a nucleotide sequence that is from 95% to 100%
identical to SEQ ID NO. 1, or its compliment, over the length of the nucleotide sequence, wherein the promoter is a constitutive promoter.

5. A promoter comprising a nucleotide sequence capable of hybridizing to the nucleotide sequence defined by SEQ ID NO:1, or its compliment, under stringent hybridization conditions comprising i) hybridization in 6 x SSC, 0.01M EDTA, 5 x Denhardt's solution, 0.5% SDS and 100µg/ml denatured salmon sperm DNA at 68°C for 3-16 hours;
ii) a first wash in 2 x SSC and 0.5% SDS at room temperature for 5 minutes;
iii) a second wash in 0.1 x SSC and 0.1% SDS at room temperature for 15 minutes;
and iv) a third wash in 0.1 x SSC and 0.5% SDS at 68°C for 2 hours, wherein the promoter is a constitutive promoter.

6. A chimeric gene construct comprising a gene for which constitutive expression is desired, and a promoter selected from any one of claim 1 to 5.

7. A vector comprising a chimeric gene construct comprising a gene for which constitutive expression is desired, and a promoter selected from any one of claim 1 to 5.

8. A method of conferring constitutive expression of a gene in a plant, comprising:
operatively linking a gene, for which constitutive expression is desired, with a constitutive promoter as defined in any one of claims 1 to 5, to produce a chimeric gene construct, and introducing the chimeric gene construct into a plant capable of expressing the chimeric gene construct.

9. A transgenic plant cell comprising the chimeric gene construct of claim 6.

10. A transgenic plant cell containing the vector of claim 7.

11. A transgenic plant cell produced using the method of claim 8.

12. A use of a promoter as defined in any one of claims 1 to 5 for directing constitutive expression of a transgene in a plant.