WO2001032897A2

WO2001032897A2 - A high level, stable, constitutive promoter element for plants

Info

Publication number: WO2001032897A2
Application number: PCT/IB2000/001601
Authority: WO
Inventors: Jan-Hendrik Groenewald; Frederik Coenraad Botha
Original assignee: South African Sugar Association; University Of Stellenbosch, Institute For Plant Biotechnology
Priority date: 1999-11-05
Filing date: 2000-11-06
Publication date: 2001-05-10
Also published as: WO2001032897A3; AU1046801A

Abstract

This invention relates to a tandem promoter construct, termed SUC-1, conferring stable, high level, constitutive expression in transgenic plants. The promoter contains both the CaMV 35S promoter and the maize UBI promoter. The promoter is particularly useful for gene expression in monocotyledonous plants, such as sugarcane.

Description

A HIGH LEVEL, STABLE, CONSTITUTIVE PROMOTER ELEMENT FOR PLANTS

BACKGROUND TO THE INVENTION

THIS invention relates to a tandem promoter construct, SUC-1 , conferring stable, high level, constitutive expression in transgenic plants.

The effective expression of transgenes in plants depends on the use of appropriate regulatory sequences. Promoter elements are the most important of these regulatory sequences. Promoter elements dictate the tissue- and developmental stage specificity and levels of gene expression. Promoters may also play a direct (Hansom et al., 1999) or indirect (Depicker and Van Montagu, 1997, Wassenegger and Pelissier, 1998) role in transgene silencing.

In addition, it has been shown that the duplication of promoter elements can lead to enhanced transgene expression (Kay et al., 1987, Omirulleh et al., 1993, Rathus et al., 1993). Similarly, the inclusion of intron sequences downstream of the promoter can lead to more efficient transgene expression (Sinibaldi and Mettler, 1992, Clancy et al., 1994). Constitutive, i.e. at all times in all tissues, transgene expression is important in all applications where a high level of the transgene product is required.

The promoter for the 35S ribozymal subunit of the cauliflower mosaic virus (CaMV) is the most commonly used promoter in transformed dicotyledonous plants (Gruber and Crosby, 1993). Unfortunately the CaMV 35S promoter is not as effective in monocotyledonous plants (Rathus et al., 1993). The most frequently used promoter in transgenic monocotyledonous plants is the maize polyubiquitin (UBI) promoter, including its δ'-untranslated and first intron sequences which, it was thought, conferred high level constitutive transgene expression (Christensen ef a/., 1992). However, more recent research has shown that endogenously the UBI promoter can not be considered constitutive (Galun and Breiman, 1997) and it does not guarantee high expression levels in all transformed monocotyledonous plants, for example in transgenic sugarcane (Hansom et al., 1999). Moreover, the UBI promoter might be directly involved in transgene silencing in sugarcane (Hansom et al., 1999).

SUMMARY OF THE INVENTION

According to one aspect of the invention a promoter comprises:

(i) a nucleotide sequence as set out in Figure 1 ;

(ii) an analogue or derivative of the nucleotide sequence as set out in Figure 1 ; (iii) a nucleotide sequence which is complementary to the nucleotide sequence of Figure 1 ; (iv) a portion of the nucleotide sequence as set out in Figure 1 ; or (v) a nucleotide sequence which hybridizes to the nucleotide sequence of Figure 1 under stringent hybridisation conditions.

The promoter is preferably a promoter for gene expression in plants, more preferably a promoter for gene expression in monocotyledonous plants.

According to another aspect of the invention an expression vector comprises a promoter of the invention located upstream of any gene to be expressed in a transgenic plant cell. An expression vector of the invention has been termed pUBI 510 (ECACC provisional accession number 00042603).

The expression vector may also comprise an endonuclease restriction site located downstream of the promoter at which a gene to be expressed in a transgenic plant cell can be inserted. According to another aspect of the invention a transformed plant cell contains a promoter of the invention or an expression vector of the invention or part thereof.

The transformed plant cell is preferably a monocotyledonous plant cell. The monocotyledonous plant cell may be derived from a plant of the Graminae family or may be derived from sugarcane.

According to another aspect of the invention a transgenic plant or plant part is provided which contains or is derived from a transformed plant cell of the invention.

The transgenic plant is preferably a monocotyledonous plant. The monocotyledonous plant may be a member of the Graminae family or may be sugarcane.

According to another aspect of the invention a method of regulating gene expression in a plant cell comprises the step of transforming the plant cell with an expression vector of the invention.

Preferably, the method involves regulating constitutive gene expression in the plant cell. More preferably the method involves regulating constitutive transgene expression in the plant cell.

The plant cell is preferably a monocotyledonous plant cell. The monocotyledonous plant cell may be derived from a plant of the Graminae family or may be derived from sugarcane. BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in more detail, by way of example only, with reference to the accompanying drawings in which

Figure 1 is the nucleotide sequence of the SUC-1 promoter element,

Figure 2 is a schematic representation of the construction of the genetic construct, pUBI 510 (ECACC provisional accession number 00042603), which can be used to clone transgenes for the direct transformation of plants,

Figure 3 is a schematic representation of the genetic construct, pGUS

512, an expression vector of the invention, in which fi- glucuronidase (GUS) expression is driven by the SUC-1 promoter,

Figure 4 shows the results obtained from a transient expression assay in sugarcane embryogenic callus using the GUS reporter gene, and

Figure 5 is a northern blot which shows the results of stable expression assays in transgenic sugarcane using a truncated Bt-toxin gene

DETAILED DESCRIPTION OF THE INVENTION

The invention involves the use of two regulatory elements in tandem to obtain a new tandem promoter construct This new tandem promoter construct has been called the SUC-1 promoter It contains both the CaMV 35S promoter and the maize UBI promoter, including the δ'-untranslated and first intron sequences The combination of these two promoters has been found to give synergistic effects It has shown high expression levels in monocotyledonous plants which are above the expression levels of both the CaMV 35S- and the UBI promoters if used alone and expression is stable

To construct the SUC-1 promoter, the maize UBI promoter including the 5'- untranslated and first intron sequences shown in Figure 1 were excised from the plasmid pAHC 20 shown in Figure 2 (Chnstensen, 1992) as a 2 0 kb fragment using the restriction enzyme Pst I The 2 Okb fragment was isolated from an agarose gel and cloned into the Pst I site of the plasmid pCFK 9, also shown in Figure 2

Thereafter the EcoR I site in the UBI intron sequence was removed by filling in the δ'-overhangs after partial digestion The resulting vector was designated pUBI 510 (ECACC provisional accession number 00042603) This is also shown in Figure 2 It contains the complete SUC-1 promoter element

Appropriate transgenes, i e sense sequences for the over expression of a particular protein or antisense sequences for the down regulation of endogenous gene expression, can be cloned into the multiple cloning site (MCS) shown in Figure 2 between the SUC-1 promoter element and the CaMV 35S termination sequence (CaMV-t) Stable transformants containing such a construct should express the recombinant gene constitutively

Transient as well as stable expression assays were done to verify the activity of the SUC-1 promoter and to compare its activity to that of known promoters

Transient assays were done using the reporter gene construct pGUS 512 shown in Figure 3 In this gene construct or vector GUS (β-glucuronidase) expression was driven by the SUC-1 promoter of the invention Transient gene expression in embryogenic sugarcane calli and young leaves was evaluated after they were bombarded with the above vector as well as with the vector pBI 221 in which GUS expression is driven by the CaMV 35S promoter and the vector pAHC27 in which GUS expression is driven by the maize UBI promoter Transformation and GUS assays were done according to standard protocols (Bower and Birch, 1992, Jefferson, 1987) In all tissues tested the SUC-1 promoter gave superior expression levels compared to the pBI 221 and pAHC27 promoters (Figure 4)

Stable expression assays were done using transgenic sugarcane plants expressing a truncated form of the CrylA endotoxin gene Transgenic sugarcane plants were produced using standard methods (Bower and Birch, 1992) Pre-embryogenic sugarcane cal were co-transformed with the pEOT 510 and pEmuKN expression vectors, allowing the selection of transgenic cells on geneticin containing media In pEOT 510 the SUC-1 promoter drives the expression of a truncated form (1 8kb) of the CrylA endotoxin gene from Bacillus thunngiensis and in pEmuKH expression of the neomicyn phosphotransferase gene (NPT II), encoding geneticin resistance, is driven by the synthetic Emu promoter sequence (Last et al , 1991) Resistant embryos were germinated and plants were regenerated on geneticin containing media Confirmed transgenic plants were hardened off and transferred to large pots for glass house trials

Mature nodes were harvested from selected clones and re-planted to obtain the equivalent of a first ratoon, i e new growth after the stalks have been harvested Transgene expression was characterised in these plants after six months to verify the stability of expression Selected transgenic plants were also exposed to drought stress to evaluate gene expression under stress conditions, because the UBI promoter is induced under certain stress conditions (Galun and Breiman, 1997) Total RNA was isolated from several transgenic lines and the CrylA mRNA quantified using a 32P labeled fragment of the CrylA gene (Figure 5). The results (lane 1 to 3) confirmed that the SUC-1 promoter gave higher stable expression levels of the transgene than the maize UBI promoter (lanes 5 and 6). The latter is especially evident under stress conditions (Figure 5).

The CaMV 35S promoter is almost silent in sugarcane. Currently, most work utilises the UBI promoter and the latter can give varying expression levels depending on the transgenic clone. Thus, the high stable expression levels achieved with the SUC-1 promoter as well as the fact that it results in strong constitutive gene expression is advantageous and completely unexpected.

DEPOSIT OF MATERIAL

The following material has been deposited with the European Collection of Cell Cultures, Centre for Applied Microbiology and Research, Salisbury, Wiltshire SP4 OJG, United Kingdom (ECACC).

Material ECACC Deposit No. Deposit Date

Plasmid pUBI510 Accession 26 April 2000 number 00042603

This deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and regulations thereunder (Budapest Treaty). REFERENCES

Bower R and Birch R: Transgenic sugarcane plants via microprojectile bombardment. The Plant Journal, 2: 409-416 (1992).

Christensen AH, Sharrock RA and Quail PH: Maize polyubiquitin genes:structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol Biol, 18: 675-689 (1992).

Clancy M, Vasil V, Hannah LC and Vasil IK: Maize Shrunken-1 intron and exon regions increase gene expression in maize protoplasts. Plant Sci, 98: 151-161 (1994).

Depicker A and Van Montagu M: Post-transc ptional gene silencing in plants. Current Op in Cell Biol, 9: 373-382 (1997).

Galun E and Breiman A: Tools for genetic transformation. In: Transgenic plants, pp44-76. Imperial Collage Press, London (1997).

Gruber MY and Crosby WL: Vectors for plant transformation. In Methods in Plant Molecular Biology and Biotechnology: 89-119 (1993).

Hansom S, Bower R, Zhang L, Potier B, Elliott A, Basnayake S, Cordeiro G, Hogarth DM, Cox M, Berding N and Birch RG: Regulation of transgene expression in sugarcane. Proc of XXIII ISSCT congress, New Delhi, India: 22-34 (1999).

Jefferson RA: Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep, 5: 387-405 (1987).

Kay R, Chan A, Daly M and McPherson: Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science, 236:1299- 1302 (1987).

Last Dl, Brettell RIS, Chamberlain DA, Chaudhury AM, Larkin PJ, Marsh EL, Peacock WJ and Dennis ES: pEmu: An improved promoter for gene expression in cereal cells. Theor Appl Genet, 82, 582-588 (1991).

Omirulleh S, Abraham M, Golovkin M, Stevanov I, Karabaev MK, Mustardy L, Morocz S and Dudits D: Activity of a chimeric promoter with the doubled CaMV 35S enhancer element in protoplast-derived cells and transgenic plans in maize. Plant Mol Biol, 21 : 415-428 (1993). Rathus C, Bower R and Birch RG: Effects of promoter, intron and enhancer elements on transient gene expression in sugarcane and carrot protoplasts. Plant Mol Biol, 23: 613-618 (1993).

Sinibaldi RM and Mettler IJ: Intron splicing and intron-mediated enhanced expression in monocots. Prog Nucleic Acis Mol Biol, 42: 229-257 (1992).

Wassenegger M and PelissierT: A model for RNA-mediated gene silencing in higher plants. Plant Mol Biol, 37: 349-362 (1998).

Claims

1 A promoter comprising

(i) a nucleotide sequence as set out in Figure 1 ,

(II) an analogue or derivative of the nucleotide sequence as set out in Figure 1 , (in) a nucleotide sequence which is complementary to the nucleotide sequence of Figure 1 , (iv) a portion of the nucleotide sequence as set out in Figure

1 , excluding a portion which encodes only the CaMV

35S promoter or a portion thereof, and excluding a portion which encodes only the UBI promoter or a portion thereof, and (v) a nucleotide sequence which hybridizes to the nucleotide sequence of Figure 1 under stringent hybridisation conditions

2 A promoter according to claim 1 for gene expression in plants

3 A promoter according to claim 2 for gene expression in monocotyledonous plants

4 An expression vector comprising a promoter as defined in any one of claims 1 to 3

5 An expression vector according to claim 4 which is pUBI 510 (ECACC provisional accession number 00042603)

6. An expression vector according to claim 4 comprising an endonuclease restriction site located downstream of the promoter at which a gene to be expressed in a transgenic plant cell can be inserted.

7. An expression vector according to claim 4 comprising a gene to be expressed in a transgenic plant cell downstream of the promoter.

8. A transformed plant cell containing a promoter as defined in claim 1 or an expression vector as defined in claim 4.

9. A transformed plant cell according to claim 8 which is a monocotyledonous plant cell.

10. A transformed plant cell according to claim 9 which is derived from a plant of the Graminae family.

11. A transformed plant cell according to claim 10 which is derived from sugarcane.

12. A transgenic plant or plant part which contains or is derived from a transformed plant cell according to claim 8.

13. A transgenic plant or plant part according to claim 12 which is a monocotyledonous plant.

14. A transgenic plant or plant part according to claim 13 which is a member of the Graminae family.

15. A transgenic plant or plant part according to claim 14 which is sugarcane.

16. A method of regulating gene expression in a plant cell comprising the step of transforming the plant cell with an expression vector as defined in claim 4.

17. A method according to claim 16 wherein the constitutive gene expression in the plant cell is regulated.

18. A method according to claim 16 wherein the constitutive transgene expression in the plant cell is regulated.

19. A method according to claim 17 or 18 wherein the plant cell is a monocotyledonous plant cell.

20. A method according to claim 19 wherein the plant cell is derived from a plant of the Graminae family.

21. A method according to claim 20 wherein the plant cell is derived from sugarcane.