WO2000023597A2

WO2000023597A2 - Tissue-specific promoters for gene expression

Info

Publication number: WO2000023597A2
Application number: PCT/GB1999/003442
Authority: WO
Inventors: Gordon Cameron Machray; Diane Davidson; Peter Edward Hedley
Original assignee: Scottish Crop Research Institute
Priority date: 1998-10-16
Filing date: 1999-10-15
Publication date: 2000-04-27
Also published as: WO2000023597A3; AU6221599A

Abstract

Expression control polynucleotides of an invertase gene are described which are tissue specific to vascular tissue and/or to nodal tissue. The expression control polynucleotides may be operably linked to a heterologous polynucleotide, for example in the form of a vector and/or used to produce transgenic plants. The heterologous polynucleotide may express polypeptides or proteins which are insecticidal or which promote or inhibit plant growth and expression thereof will be localised to the vascular tissue. Where the transgenic plant is a potato plant, stolon formation and/or tuber sprouting may be likewise controlled through localised expression of suitable heterologous gene products in nodal tissue.

Description

TISSUE-SPECIFIC PROMOTERS FOR GENE EXPRESSION

This invention relates to the fields of plant biotechnology and plant genetic engineering. In particular the invention relates to transgenic plant production and tissue-specific expression of heterologous gene sequences in plants.

A promoter is a non-coding nucleotide sequence which controls the transcription of an adjacent nucleotide sequence. A number of promoters have been isolated from a wide variety of sources, including plants. In certain applications it is desirable to genetically engineer a construct which comprises a promoter operatively linked to a heterologous (non-native) nucleotide sequence such that the promoter controls expression of the heterologous sequence in the host cell transformed with that construct. Where the promoter is only active in particular tissue types expression of the heterologous sequence is restricted accordingly and this may be especially desirable in some circumstances.

Expression of heterologous genes may render the plant resistant to pest or pathogen attack, for example if the heterologous gene encodes a protein toxic to the pest or which inhibits the normal processes of infection by the pathogen. Tissue-specific promoters can also be used to activate the expression of heterologous genes encoding proteins which will enhance the nutritional value of the specific tissues where the promoter is active. Such promoters may also be used to modify the level of endogenous gene products in the specific tissues concerned, thereby modulating the biochemistry, physiology or development of the plant.

The number of tissue-specific promoters which have been well characterised is limited and different promoters exhibit a range of activities which cannot be predicted a priori and are difficult to quantify. The activity of a promoter isolated from one species of plant may also differ when it is utilised in a heterologous species - such differences may be both in the tissue specificity and strength of the promoter and are more likely to occur with greater taxonomic distance between plant species. In addition different promoters may be required to control expression of multiple genes since gene silencing effects can occur if duplicate copies of the same promoter are used. The choice of promoter is therefore limited and has to be experimentally verified for each system under study.

In our earlier WO-A-98/41643 we describe an invertase gene expression control polynucleotide which is pollen- cell specific. We have now located further invertase gene expression control polynucleotides which are cell- specific to other plant cell types.

According to the present invention there is provided an invertase gene expression control polynucleotide, a derivative, a functional equivalent, or a part thereof, which is cell-specific to vascular tissue or to nodal tissue .

By "cell-specific to vascular tissue" we mean that the expression control polynucleotide exhibits a distinct level of activity (or lack of activity) in cells of vascular tissue compared to the other tissue types of the transformed plant.

By "cell-specific to nodal tissue" we mean that the expression control polyncleotide exhibits a distinct level of activity (or lack of activity) in cells of the node between the stem and the petiole (including the equivalent "eyes" of tubers) compared to other tissue types of the transformed plant .

By "expression control polynucleotide" we mean any polynucleotide which is capable of affecting the expression of a gene. The term is intended to include promoters, enhancers and suppressors.

By "functional equivalent" we mean any variation of the expression control polynucleotide which exhibits substantially the same functional properties of the original polynucleotide.

By "derivative" we mean a modified version of the expression control polynucleotide which exhibits substantial sequence homology (for example 70% sequence homology) to the original polynucleotide, for example which include nucleotide substitutions which have no effect on biological function.

By "part" we mean a deleted version of the expression control polynucleotide, which comprises at least a substantial portion of the original polynucleotide (for example at least 50%, more usually 70%, for example 80% or even 85 to 90%, of said polynucleotide) .

The preferred type of expression control polynucleotide is a promoter.

The invertase gene promoter is preferably derived from a dicotyledon, especially a member of the Solanaceae, for example potato.

The expression control polynucleotide of the invention may comprise double- or single-stranded DNA or RNA.

The invention also provides the use of the expression control polynucleotide described above to control expression of heterologous sequences. Optionally the expression control polynucleotide is used to drive cell -specific expression of protein-encoding heterologous genes in plants eg monocotyledons or dicotyledons. Use of the expression control polynucleotide in this way in dicotyledons is preferred.

The invention also provides a recombinant expression control polynucleotide comprising at least a part of a cell -specific expression control polynucleotide as described above. The recombinant expression control polynucleotide of the invention is capable of expression of a heterologous sequence in specific cells. The heterologous sequence expressed may encode a protein. Alternatively RNA sequences which do not code for protein (eg ribosomal RNA or anti-sense RNA) may instead be transcribed from the heterologous sequence.

The invention also provides a polynucleotide having the sequence set out in SEQ ID Nos 2, 3 and 4, including derivatives, functional equivalents or parts thereof.

A deposit of the plasmids pGE-GUS, pCDl-GUS and pCD4- GUS containing preferred expression control polynucleotides has been made on 7 October 1998 at the National Collection of Type Cultures under Nos NCTC 13117, NCTC 13118 and NCTC 13119 respectively.

The present invention also provides a recombinant polynucleotide construct comprising an expression control polynucleotide according to the invention. The construct may be operably linked to a heterologous (preferably protein-encoding) polynucleotide.

Thus, activation of the expression control polynucleotide may drive the expression of the heterologous polynucleotide, enabling production of the encoded protein. Since the expression control polynucleotide is tissue-specific, production of the protein will be limited to those tissues where the expression control polynucleotide is active.

The present invention also provides a recombinant vector containing an expression control polynucleotide or a recombinant polynucleotide construct as defined above .

According to the present invention there is also provided a method of producing a recombinant vector, said method comprising ligating an expression control polynucleotide as described above into a suitable vector. A method of producing a transformed cell by transfecting a host cell using said recombinant vector forms another aspect of the invention. Suitable vectors and genetic modifications thereof are well- known in the art. The present invention also provides a transformed host cell containing a recombinant polynucleotide construct or vector as defined above.

The present invention also provides a transgenic organism (for example a transgenic plant) containing a recombinant polynucleotide construct or a vector as defined above. The progeny (and seeds or tubers) of such transgenic organisms forms a further part of the invention.

The present invention also provides a method for controlling the expression of a protein, said method comprising operably linking a polynucleotide sequence encoding said protein to an expression control polynucleotide of the invention. The method is especially useful for the expression of proteins in vascular tissue, for example to suppress or promote budding of potato tubers; to develop "bushier" or non- branching varieties of tomato plants; to express an insecticide (since vascular tissue is a target site for all sap-sucking insects) ; or to promote or reduce growth of the plant (i.e. to obtain taller or dwarf varieties of plants) .

Thus the invention also provides a method of controlling the expression of a heterologous polynucleotide in vascular tissue, said method comprising operably linking said heterologous polynucleotide to an expression control polynucleotide of the invention.

In one embodiment the invertase gene promoter described in pGE-GUS is expressed in potatoes to delay or promote stolon formation (i.e. to obtain "early" or "late" varieties of potatoes) . Alternatively the promoter could be expressed in potatoes to delay or promote potato tuber sprouting (i.e. to allow for prolonged storage or to initiate potato plant growth) . In particular the present invention encompasses the tubers of such transgenic potato plants.

In an alternative embodiment the invertase gene promoter described in pCDl-GUS or pCD4-GUS is expressed in a plant (especially a plant of the species of the Solanaceae, for example potatoes or tomatoes) to express an insecticide or insect repelling compound or to express a protein to promote or inhibit plant growth.

The present invention will now be further described with reference to the non-limiting Example and accompanying Figures in which:

Fig. 1: GUS staining of stems of transgenic potato plants containing the invGE promoter fragment fused to uidA .

Fig. 2: GUS staining of tuber (under eyes) of transgenic potato plants containing the invGE promoter fragment fused to uidA.

Fig. 3: GUS staining of stems of transgenic potato plants containing the invCDl promoter fragment fused to uidA in longitudinal (A) and cross (B) section.

Fig. 4: GUS staining of stems of transgenic potato plants containing the invCD4 promoter fragment fused to uidA in longitudinal (A) and cross (B) section. EXAMPLE

A potato (Solanum tuberosum L . ) cv. Saturna genomic library, consisting of a partial Sau3AI digest of genomic DNA cloned into XEMBL3 , was plated to yield 1 x 10⁵ pfu which were screened with a radiolabelled carrot invertase cDNA fragment generated by reverse transcription-polymerase chain reaction (RT-PCR) using primers derived from a sequence of carrot cDNA (Sturm and Chrispeels, 1990, The Plant Cell, 2:1107-1119). Hybridisation conditions of 5xSSC at 65 °C were utilised with subsequent low stringency washing of filters in 2xSSC at 65 °C. After three rounds of screening two positive clones were obtained plaque pure. DNA was purified from one positive clone XGF5 , which was shown to contain an insert of approximately 23 kb of potato DNA. This cloned potato DNA was digested with Xfoal and Sail , and fragments cloned into pUC19. One subclone, named pGF521, contained 5.4 kb of the potato DNA. A complete DNA sequence of this fragment is presented (SEQ ID No 1) . It was determined, by homology to known invertase gene sequences, that the 5.4 kb of potato DNA carried partial sequences of two invertase genes with the intergenic region constituting the promoter of the downstream gene. This promoter (invGF) forms the basis of a previous patent application (WO 98/41643) .

The upstream gene (invGE, bp 1-3407) was incomplete in this clone since the 5' end of the clone terminated within intron I and lacked exon I and promoter sequences further upstream. In order to acquire 5' sequence of invGE an approach involving nested direct PCR followed by nested inverse PCR was used. Firstly, using potato cv. Saturna DNA as template and primers based on known sequences (Tables 1 and 2), nested PCR generated a DNA fragment of 590 bp which, on cloning and sequencing, was shown to include the remainder of intron I and 50 bp of exon I of invGE .

m=a+c k=g+t y=c+t

Then primers derived from this new sequence (Tables 3 and 4) were used on cv. Saturna DNA, which had been BamHI -digested and religated, in a nested inverse PCR approach to generate a product of <1 kb which, on cloning and sequencing, was shown to complete exon I and include 774 bp of DNA upstream of the start codon of this gene (SEQ ID No 2) . At each stage an overlap of approx. 200 bp of identical sequence between the newly-generated and existing sequence information confirmed the identity of their origin in the potato genome, and the contiguity of this sequence was confirmed by direct PCR from the genome, employing a primer from the 5' end of the IPCR-generated sequence and a second primer from the lambda clone, which generated the expected size and sequence of product.

A 794 bp fragment from the BarriH.1 site at the 5' end of the promoter sequence to an Alul site 20 bp into the first exon of invGE was isolated, BamHI linkers added, and after digestion with BamHI, the fragment was cloned into pBIlOl to give plasmid pGE-GUS which was used as a vector for stable plant transformation. In pGE-GUS the promoter fragment is fused to the uidA gene from Escheri chia coli and, when the promoter is active in plants, drives the transcription of this gene to produce the bacterial enzyme /3-glucuronidase (GUS) . A series of transgenic lines of potato (cv. Desiree) plants was generated by Agrobacterium tumefaciens- mediated transformation using pGE-GUS as a vector. Plants derived from the use of pGE-GUS as a vector were passed through one cycle of tuberisation then grown in a controlled environment until flowering occurred. A range of tissues including leaf, stem, root, tuber and floral tissues were analysed by a GUS histochemical assay to detect the activity derived form the uidA gene activated by the invertase promoter. Blue staining, indicative of GUS activity, (appearing in the Figures as more darkly shaded areas) was observed at the adaxial side of petioles at nodes in the stem where axillary bud primordia are sited (Figure 1) and extending into the neighbouring vascular tissue. Blue staining was also observed under the "eyes" of tubers (Figure 2), that is at areas underlying shoot primordia in resting tubers which also contain vascular tissue servicing the shoot.

Previous screening of a potato cv. Cara cDNA library had identified two cDNA clones encoding further potato inverses (pCDlll and pCD141; Hedley et al . , 1994, Gene, 145:211-214). Promoter sequences of both genes encoding these cDNAs were also obtained using a nested inverse PCR approach. Using potato cv. Cara DNA which had been BcoRI -digested and religated as template and two sets of primers derived form the cDNA of pCDlll (Tables 5 and 6) and used in sequential PCRs, a further 961 bp of promoter sequence (SEQ ID No 3) 5' to this gene (invCDlll) was amplified, cloned and sequenced.

In a further PCR bp 9-916 of this sequence, constituting the invCDlll promoter sequence used further and designated CD1, was amplified using primers containing BarriRI and HindiII sites. After digestion with _3a_nHI and Hindi11 the purified PCR product was cloned into similarly-digested pBIlOl to generate plasmid pCDl-GUS.

Using potato cv. Cara DNA which had been Hindlll- digested and religated as template and two sets of primers derived from the cDNA of pCD141 (Tables 7 and 8) and used in sequential PCRs, a further 1421 bp of promoter sequence (SEQ ID No 4) 5' to this gene (invCD141 ) was amplified, cloned and sequenced.

In a further PCR bp 28-1421 of this sequence, constituting the invCD141 promoter sequence used further and designated CD4 , was amplified using primers containing BaπiH.1 and Hindi11 sited. After digestion with BamHI and HindiI I the purified PCR product was cloned into similarly-digested pBIlOl to generate plasmid pCD4-GUS.

Plasmids pCDl-GUS and pCD4-GUS were used as vectors for stable plant transformation. In both plasmids the promoter fragment is fused to the uidA gene from Escheri chia coli and when the promoter is active in plants would drive the transcription of this gene to produce the bacterial enzyme 3-glucuronidase (GUS) . A series of transgenic lines of potato (cv. Desiree) plants was generated by Agrobacterium tuwefaciens- mediated transformation using pCDl-GUS as a vector and a second series using pCD4-GUS as a vector. Transgenic plants derived from their use as a vector were passed through one cycle of tuberisation then grown in a controlled environment. A range of tissues including leaf, stem, root and floral tissues were analysed by a GUS histochemical assay to detect the activity derived from the uidA gene activated by the invertase promoters.

In CD1-GUS transgenic plants strong blue staining was observed in the stem of the plant extending into the petioles and base of the major vein of leaves (Figures 3a and b) . This staining was consistent with staining in both large and small vascular bundles. A similar pattern of staining was observed in CD4-GUS transgenic plants (Figures 4a and b) but the staining was less intense indicating a possible lower level of activity of this promoter. Detailed histochemical analysis of the staining region from both sets of transgenics confirmed that the staining was localised in the phloem elements of the vascular bundles. The recombinant DNA procedures utilised were as described by Sambrook et al . , 1989, Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Plant tissue culture and transformation protocols were as detailed by Hedley, 1995, PhD thesis, University of Dundee, United Kingdom. Histochemical assay of GUS was performed as indicated by Jefferson, 1987, Plant Molecular Biology Reporter, 5:387-405.

Claims

1. A vascular tissue or nodal tissue cell-specific expression control polynucleotide of an invertase gene, or a derivative, functional equivalent or part of said polynucleotide.

2. An expression control polynucleotide as claimed in Claim 1 which is a promoter.

3. An expression control polynucleotide as claimed in either one of Claims 1 and 2, which comprises a sequence substantially as set out in SEQ ID No 2 or as present in NCTC 13117, or a derivative, functional equivalent or part of said polynucleotide.

4. An expression control polynucleotide as claimed in either one of Claims 1 and 2, which comprises a sequence substantially as set out in SEQ ID No 3 or as present in NCTC 13118, or a derivative, functional equivalent or part of said polynucleotide.

5. An expression control polynucleotide as claimed in either one of Claims 1 and 2, which comprises a sequence substantially as set out in SEQ ID No 4 or as present in NCTC 13119, or a derivative, functional equivalent or part of said polynucleotide.

6. A recombinant expression control polynucleotide comprising at least a part of a polynucleotide as claimed in any one of Claims 1 to 5.

7. A recombinant polynucleotide construct which comprises at least part of an expression control polynucleotide as claimed in any one of Claims 1 to 5, and which is in the form of a vector.

8. A recombinant polynucleotide construct which comprises at least part of an expression control polynucleotide as claimed in any one of Claims 1 to 5 operably linked to a heterologous polynucleotide.

9. A recombinant polynucleotide construct as claimed in Claim 8 in the form of a vector.

10. A recombinant polynucleotide construct as claimed in either one of Claims 8 and 9 wherein said heterologous polynucleotide encodes a polypeptide or protein.

11. A host cell transformed with a recombinant polynucleotide as claimed in Claim 6 or with a construct as claimed in any one of Claims 7 to 10.

12. A plant host cell as claimed in Claim 11.

13. A transgenic non-human organism transformed with a recombinant polynucleotide as claimed in Claim 6 or with a construct as claimed in any one of Claims 7 to 10.

14. An organism as claimed in Claim 13 which is a plant.

15. An organism as claimed in Claim 14 which is a member of the Solanaceae.

16. An organism as claimed in Claim 15 which is a potato or tomato plant.

17. An organism as claimed in any one of Claims 13 to 16 wherein said expression control polynucleotide is vascular tissue cell-specific and has a heterologous protein-encoding polynucleotide operably linked thereto, said heterologous polynucleotide encoding a polypeptide or protein which has insecticidal or insect repelling properties.

18. An organism as claimed in any one of Claims 13 to 16 wherein said expression control polynucleotide is vascular tissue cell-specific and has a heterologous protein-encoding polynucleotide operably linked thereto, said heterologous polynucleotide encoding a polypeptide or protein which promotes or inhibits plant growth.

19. An organism as claimed in any one of Claims 13 to 16 wherein said expression control polynucleotide is nodal tissue cell-specific and has a heterologous protein-encoding polynucleotide operably linked thereto, said heterologous polynucleotide encoding a polypeptide or protein which delays or promotes stolon formation.

20. A potato plant as claimed in any one of Claims 13 to 16 wherein said expression control polynucleotide is nodal tissue cell -specific and has a heterologous protein-encoding polynucleotide operably linked thereto, said heterologous polynucleotide encoding a polypeptide or protein which delays or promotes potato tuber sprouting.

21. Potato tubers obtained from a plant as claimed in Claim 20