DE19730535A1 - Special viral DNA fragments and their use as promoter - Google Patents

Abstract

The invention relates to the characterization and use of powerful viral promoters for expressing genes, especially in plants. The invention is based on the surprising discovery that special DNA fragments derived from CFDV-DNA (coconut foliar decay virus DNA), which feature the stem-loop structure but not the translation start(s) for the open reading frames ORF1 and/or ORF2, have a distinctly higher promoter activity in plants than promoters derived from CFDV-DNA which do not correspond to these requirements.

Description

It is well known that by means of genetic engineering scher work techniques targeted individual genes in the genome of Living organisms, such as microorganisms, yeasts or plants, transferred to let. These as a transformation or higher cells too Technique known as transfection is routinely applied to ver carried out various ways, z. B. by "particle gun bom bardment "(see M. E. Fromm, F. Morrish, C. Armstrong, R. Wil liams, J. Thomas and T.M. Klein: "Inheritance and expression of chimeric genes in the progeny of transgenic maize plants ", Bio / Technology 8: 833-839, 1990), "naked DNA transfer" (cf. P. Meyer, I. Heidmann, G. Forkmann and H. Saedler: "A new petu nia flower colored by transformation of a mutant with a maize gene ", Nature 330: 677-678, 1987) or by Agrobacte rium-mediated stable integration of genes or genab cut into the genome of a recipient plant. As alternative for the chromosomal integration of foreign genes can, for. Ex trachromosomally replicating vectors are used to Foreign genes without integration into a desired organism too express. For plants, for example, ex Trachromosomal replicating vectors are available that are made Plant viruses have been developed (see, for example, J.W. Davies and J. Stanley: "Geminivirus Genes and Vectors", Trends Genet. 5: 77-81, 1989). The expressing in the chosen organisms Foreign genes must be under the control of for this purpose organism suitable regulatory signals (promoter, Terminator), which are either for a konstituti ve, tissue and / or developmental transcription sor In addition, it is desirable to use a  strong promoter to increased mRNA synthesis of the foreign gene effect.

A well-known promoter for plants that meets the requirement fulfilled a strong, constitutive promoter and therefore before mainly used for plant transformation (see R. Walden: "Genetic Transformation in Plants", Open University Press, Milton Keynes, 1988) is the 35S RNA promoter of cau Liflower mosaic virus (CaMV).

A disadvantage of the known CaMV 35S promoter is its low activity in monocotyledons and in phloem tissue.

In the German Patent DE 43 06 832 of the Max Planck Society for the Advancement of Science and in Rohde et al., Plant Molecular Biology 27: 623-628, 1995, was the use of a virus CFDV from the cococnut Cocos nucifera (Coconut foliar decay virus) originating DNA whose structure is shown in FIGS . 1, 3A and 3B of the patent, as a viral phloem-specific promoter for tissue-specific expression of genes in transgenic plants be written.

The CFDV virus is in the vascular system of the plant loka (see J.W. Randles et al .: "Localization of coconut foliar decay virus in coconut palm ", Ann. Appl. Biology 1992, 601-617). One with the disease symptoms and the occurrence viral particle-associated DNA was previously cloned have been sequenced and their structure determined (see W. Rohde et al .: "Nucleotide sequence of a circular single-stranded DNA as sociated with coconut foliar decay virus ", Virology 176: 648-651, 1990). CFDV is a viral phytopathogen with a genome from single-stranded, circular, covalently closed DNA. In Rohde et al., Virology 176: 648-651, 1990, a DNA Molecule of the CFDV with a size of 1291 nucleotides and De letion mutants thereof described. CFDV is not a representative of Geminivirus group, but probably forms the prototype of DNA virus group of the "circoviruses".  

For the phloem specificity of this virus could be different Be responsible for mechanisms. For PLRV (potato leafroll vi rus) z. B., a representative of the luteovirus group, ge show that a suppressor tRNA, to which the virus for its gene expression is dependent, is present only in the phloem and thereby virus propagation beyond this tissue ver hinders (Rohde et al., unpublished data).

The object of the invention is compared to the genann th promoters to provide stronger promoter, which in particular for the tissue-specific expression of genes in transgenic plants and both monocotyledons and is also active in dicotyledons as well as in phloem tissue.

It was found that the task with special len virus DNA fragments can be released by the DNA of the CFDV virus in the manner indicated in claim 1 tet are.

The invention therefore relates to the An spells labeled virus DNA fragments and their use as promoters.

Surprisingly, it was found that the soge called "stem-loop" structure, which is generally considered as necessary Element for the replication of CFDV and geminiviruses will see a decisive influence on transcription exercises. Thus, constructs for the transient expression of a Reporter gene in potato protoplasts only active when the "stem-loop" structure is obtained. In addition, gefun was that the presence of the translation start (s) for the two open CFDV reading ORF1 and / or ORF2 the Translation of a reporter gene negatively affected.

The CFDV fragments according to the invention are dement speaking through the complete "stem-loop" structure and the Absence of the translation start (s) for the open Le seraster ORF1 and / or ORF2 of the CFDV.

Relative to the 5'-end of the cleavage of the circuli DNA of the CFDV with the restriction endonuclease XhoI resul  linearized DNA as position 1 comprises the stem-loop Structure nucleotides 941 to 971; the open CFDV Reading frame ORF1 and ORF2 comprise nucleotides 1004 to 583 or 1215-383.

For the preparation of inventive CFDV DNA fragments be one is familiar to those skilled in well-known techniques, such as For example, suitable restriction endonucleases clones on the CFDV DNA or polymerase chain reaction technology that makes it possible, by means of specific primers CFDV- DNA fragments of the desired length starting from a full length gene to amplify CFDV DNA construct. For this purpose are on known manner, the primer according to the desired CFDV fragment using the methods described by W. Rohde et al. in Virology 176: 648-651, 1990 described nucleotide sequence of the CFDV virus and more precisely, the nucleotide sequences in the region of the 5 'and 3' ends, respectively the desired fragment synthesized.

Particularly preferred CFDV DNA Frag according to the invention mente are the DNA fragments with the nucleotides 211 to 991, 409 to 991, 611 to 991 or 711 to 991.

In comparison to those in German Patent DE 43 06 832 described promoters have the new constructs überra up to a 4-fold increase in activity, in the Compared to the CaMV 35S promoter one up to 2-fold higher Activity in plant cells. In particular, a strong and specific expression of genes under control observed these promoters according to the invention in phloem tissue. Accordingly, an important scope of the invention For example, the phloem-specific expression of luteo viral genes with the aim of creating virus-resistant plants fen. Luteoviruses such. B. PLRV (potato leafroll virus) are phloem-specific replicating viruses and the previously u. a. ver CaMV 35S promoter showed only weak activity in the Phloem.  

Another surprising finding is the fact that CFDV DNA fragments according to the invention also in bacteria have significantly higher activity than that also in Bak active CaMV 35S promoter (Assaad and Signer, Molecular and General Genetics 223: 517-520, 1990). Thus, the CFDV Construct pRT CF4 up to 60-fold higher activity in E. coli as the CaMV 35S promoter. Because of this activity The CFDV promoters of the invention are also suitable for the Use in bacterial systems, for example for the manufacture development of pharmacologically active proteins or peptides.

Further investigations indicate an equally high level Activity of these CFDV fragment promoters in yeasts and fungi out.

Likewise subject of the invention are DNA fragments, those of the CFDV fragments defined above by Sub institution, deletion, insertion or modification of individual Nucleotides or smaller groups of nucleotides derived are and that have a comparable promoter activity as the off and their use as Promoto ren. As a comparable promoter activity, for example a promoter activity that is up to 20% over or below that of the starting fragment.

Further objects of the invention are transformed Plant, bacterial and yeast cells produced using the DNA fragments according to the invention were obtained.

The figures show in:

Fig. 1: the schematic structure of the CFDV DNA with 6 possible open reading frames (ORF1-6) and the so-called "stem-loop" structure. The arrow points to the XhoI interface.

Fig. 2: the so-called "stem-loop"structure; it shows homology to a similar structure in the genome of geminiviruses and is believed to be responsible for the replication of the virus.

FIG. 3 shows the schematic arrangement of the possible signals for the transcription regulation of the linearized by cutting at the Xho I site CFDV DNA. The arrows indicate the larger open reading frames ORF1, ORF2, ORF3 and ORF4 on the CFDV DNA. The location marked TATAA comprises a possible TATA box, the abbreviation RPT associated with two arrowheads indicates a repeated sequence; the stem-loop structure is labeled SL.

FIG. 4 shows the sequence of the two repeated sequences (RPT) and their arrangement as a stable "stem-loop" - structures with the usual CGAAG-loop sequence.

Fig. 5: a schematic representation of the position of different CFDV fragments used for constructs for determining the promoter strength on the CFDV DNA linearized by section at the XhoI site. The arrowheads indicate the location of the two directly repeated sequences (RPT) above a 52 bp element (black box). This element shows 70% sequence identity between CoYMV and CFDV. The arrows indicate larger open reading frames in the three reading frames 1 , 2 and 3 (ORF1, ORF2, ORF3) of the CFDV DNA. The abbreviation TATAA indicates a possible TATA box, the location of the "stem-loop" structure is also indicated. XboI, AflIII and StyI characterize the location of restriction endonuclease sites.

From the schematically represented CFDV fragment Promoters are those with pRT CF2, pRT CF3, pRT CF4 and pRT CF5 designated fragments inventin DNA fragments of the invention. For comparison purposes listed are not the invention CFDV constructs pRT CF7, pRT CF8, pRT CF9 and pRT CF10, while still all the TATAA box but at the 3 'end of the CFDV sequence are deleted so that a training of "stem-loop" structure is no longer possible. Likewise for comparative purposes serve that in the German Patent P 43 06 832, not construct according to the invention pRT Xho / Sty, which the Translation start of the ORF1 open reading frame with, and denoted by 35S accordingly close CaMV 35S construct.

FIG. 6 shows the schematic structure of the starting pRTsynLUC.

Investigations on the promoter strength of different CFDV fragments in plants and bacteria

For investigations on the promoter region and strength by the transient expression in plant cells and bacteria wur the fragments of CFDV DNA from a CFDV construct full length (Rohde et al., Plant Mol. Biol. 27: 623-628, 1995) first by the polymerase chain reaction (PCR) amplifi and as subgenomic fragments in the plasmid vector pRT2synGUSΔH into transcriptional fusion to the gene of β- Glucuronidase (GUS) brought. The resulting plasmids were in Experiments on transient expression with a correspond  the CaMV 35S construct as well as with constructs not invented Compared to the invention CFDV DNA fragments.

All of the process steps listed below were unless otherwise stated, according to standard ver as described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA (1989). executed.

example I. Preparation of the CFDV fragment-GUS constructs for the transient expression

The starting plasmid was the full length CFDV construct Biol. 27: 623-628, 1995 was written. With the help of specific primers, the additional Restriktionsschnittstellen contained, HindIII for the 5'-end and NcoI for the 3'-end of the amplified DNA Molecules, the CFDV genome was amplified. According to the Choice of primers were fixed in terms of their length CFDV fragments obtained. The primers were determined on the basis of W. Rohde et al. in Virology 176: 648-651, 1990. Nu Keitelfidsequenz of the CFDV virus and more precisely on the Nu kleotidsequenzen in the range of 5, or 3 'ends of the desired fragment to be synthesized by the later DNA Amplification listed in Table 1 below To gain CFDV fragments. The primers were in addition to DNA Sections supplemented the above additional Contained restriction sites.

The amplification products were with HindIII / NcoI ver daut, the cleavage products separated in an agarose gel and isolated the desired DNA fragments by electroelution.

The CFDV fragments were then ligated into the vector pRT2synGUSΔH prepared in advance from the plasmid pRTsynLUC ( Figure 6, Turner et al., Arch. Virol 137: 123-132, 1994). For this purpose, the luciferase gene was removed by NcoI / BamHI digestion and replaced by the GUS gene with NcoI / BamHI ends. Finally, the Hindiil site was deleted at the 35S 3 'end by partially cleaving the plasmid with HindIII, filling in the site, and circularizing the linear molecule to pRT2synGUSΔH. Instead of the HindIII site, an NheI site was created. The 35S promoter was removed from this plasmid by HindIII / Ncol digestion and replaced with the corresponding HindIII / NcoI-CFDV fragments.

The CFDV fragments contained as promoters in the generated CFDV fragment-GUS constructs are listed in Table 1 with regard to their exact position on the CFDV DNA and are shown schematically in FIG . The nucleotide positions indicated in Table 1 relate to a CFDV DNA linearized by cleavage with the restriction endonuclease XhoI, whose 5'-end has been assigned position 1. In addition, the corresponding DNA sections for the stem-loop structure, the ORF1 and ORF2 open reading frames, and other structural elements of the CFDV DNA were also included.

The CFDV fragments which are shown in Table 1 and are shown schematically in FIG. 4 and designated pRT CF2-5 are CFDV fragments according to the invention. The CFDV fragments recorded with pRT CF7-10 are fragments of CFDV not according to the invention, since they still comprise the TATAA box, but in this case the CFDV sequence is so deleted at its 3 'end that a formation of the "stem-loop" structure is no longer possible.

TABLE 1

The Kon also used for comparison purposes pRT CF XS and pRT 35S, which express the GUS reporter gene in Ver binding with the XhoI / StyI fragment of CFDV virus (Table I) or the CaMV 35S promoter were, as in the German Patent P 43 06 832, prepared.  

II. Transient expression of CFDV fragment-GUS constructs in Tobacco protoplasts II.1. Protoplast media

K3: Macro elements microelements 25 mM KNO ₃ 100 μM H ₃ BO ₃ 1 mM NaH ₂ PO ₄ 130 μM MnSO ₄ 6 mM CaCl ₂ 40 μM ZnSO ₄ 3 mM NH ₄ NO ₃ 5 μM KCl 1 mM (NH ₄ ) ₂ SO ₄ 1 μM CuSO ₄ 1 mM MgSO ₄ 1 μM CoCl ₂

Iron in EDTA vitamin solution 1 μM FeSO ₄ 270 μM glycine 1 μM Na ₂ EDTA 160 μM nicotinic acid 100 μM pyridoxine 3 μM thiamine

carbohydrates hormones 400 mM sucrose 5.5 μM NAA 1.7 mM xylose 1.0 μM kinetin 0.5 mM inositol @ pH 5.6 osmotic value: 600 mOs

W5:
150 mM NaCl
125 mM CaCl ₂

5mM KCl
5mM glucose
pH 5.6-6.0

MaMg:
450 mM mannitol
15 mM MgCl ₂
0.1% MES
pH 5.6

II.2. Producing tobacco protoplasts

(See I. Negrutiu et al., "Fusion of plant proto plasts: a study using auxotrophic mutants of Nicotia na plumbagenifolia, viviani ", Theor. Appl. Genet. 72: 279-286, 1987).

Leaves (10 g) of Nicotia na tabacum plants grown in tissue culture (var. SR1) were incubated in 100 ml of enzyme solution for 16 h at 25 ° C in the dark and the resulting protoplasts separated by sieves (mesh size 100 microns) of coarse tissue remnants , The further purification of the protoplasts was carried out by repeated centrifugations and washing with K3 medium, wherein the vital protoplasts each concentrated on the surface, and finally by resuspension in W5 medium and sedimentation by centrifugation. The protoplasts sediment was taken up in MaMg buffer and adjusted to a concentration of 10 ⁶ / ml.

II.3. Transformation of protoplasts

(see C. Maas and W. Werr: "Mechanism and optimized conditions for PEG mediated DNA transfection into plant protoplasts ", Plant Cell Rep. 8: 148-151, 1989).

To each 500 .mu.l protoplasts were added 15 .mu.l plasmid / Carrier DNA (corresponding to 10 ug CFDV fragment-GUS construct or CaMV35S-GUS plasmid DNA and 50 ug calf thymus DNA), the suspension for 10 min at Incubated at room temperature, then carefully with PEG solution (40% PEG 4000, 0.1 M Ca (NO ₃ ) ₂ , 0.4 M mannitol) and immediately swirled until no more streaks were visible. After a further 30 minutes of incubation, the addition of 4 ml of K3 medium (with antibiotic and kinetins) was carried out and the individual transformation mixtures were kept in the dark for 20 h at 25 ° C.

II.4. Analysis of protoplast transformations

The protoplast attachments were after 20 h with W5 medium on 10 ml filled, centrifuged, the sedimented Protopla after centrifugation in 1 ml of W5 medium again centrifuged and frozen in liquid nitrogen. For the determination of Protein amount and GUS enzyme activity were mortared the Protopla in 50 μl of GUS extraction buffer and certain of the CIS Activity fluorimetric with 4-methylumbelliferyl-β-D-glucu ronid (4-MUG, see R. A. Jefferson: "Assaying chimeric genes in plants: the GUS gene fusion system ", Plant Mol. Biol. Rep. 5: 387-405, 1987). For this purpose, 4-methylumbelliferyl-β-D- glucuronide (4-MUG) for 1 h at 37 ° C. The amount of protein was determined to Bradford (see M. Bradford: "A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding Anal. Biochem. 72: 248-254, 1976).

The results obtained for each construct are listed in Table 2 below. The results in Table 2 is given as the percentage of activity of the individual CFDV constructs related to that of the CaMV 35S Promoter construct (pRT 35S), which was set to 100%. It will be the results of two or three independent Experi ment as well as the mean of these results ben. The construct pRT CF XS contains the in the German Pa Tent 43 06 832 disclosed, non-inventive fragment, by the cleavage of the CFDV DNA by means of the restriction XhoI and StyI is obtained and in addition to the donukleasen Translation start of the ORF1 open reading frame comprises.  

TABLE 2

As the results listed in Table 2 show, white sen the CFDV fragments according to the invention a significantly higher Promoter activity in tobacco protoplasts as the XhoI / Styl CFDV Fragment promoter of the construct pRT CF XS, which in addition contains the translation start of the ORF1 open reading frame and in German Patent P 43 06 832 has been described.

The non-inventive constructs pRT CF7-10 show in tobacco protoplasts no activity, indicating that the Possibility of forming the "stem-loop" structure in the area nucleotides 941 to 971 in the CFDV fragment promoter for the promoter activity is essential.

The construct according to the invention pRT CF4 shows in tobacco Protoplasts also have a comparable promoter activity such as the CaMV 35S promoter (see construct pRT 35S).  

III. Transient expression of CFDV fragment-GUS constructs in E. coli III.1. Transformation of E. coli

Competent E. coli JM109 cells were treated with the corre sponding transformed plasmid DNAs by electroporation and on LB plates (with the addition of ampicillin) selected.

III.2. Analysis of E. coli transformations

Single colonies were allowed to grow up in 2 ml of LB medium (with the addition of ampicillin) overnight. 10 .mu.l of bacterial suspension were digested with 35 .mu.l extraction buffer (50 mM sodium phosphate buffer, pH 7, 10 mM EDTA, 0.1% Triton X-100), with 5 .mu.l 10x 4 MUG solution (4-methylumbelliferyl-β Biol. Rep. 5: 387-405, 1987) and for 10 min, 20 min, or at 37 ° C for 10 min or to measure the time course of GUS activity Incubated at 37 ° C for 47 min. The reaction was stopped by addition of 1 ml of 0.2 M Na ₂ CO ₃ buffer and the GUS activity was determined by 4 MUG fluorometry. The amount of protein was determined by Bradford (see M. Bradford, Anal Biochem., 72: 248-254, 1976).

The results obtained for each construct are listed in the following Tables 3A and 3B. The results The values in Table 3A are given as a percentage of activity based on the activity of the CFDV promoter construct pRT CF4, which is the highest overall Pro achieved in this example motor activity was set to 100%. It will be the results of two or three independent experiments as well as the means value given from these results. The in Table 3B in the each right column for the individual incubation periods given percentages indicate the percentage of activity be referred to that of the CFDV promoter construct pRT CF4 ent  speaking the absolute values listed in the respective left columns for selected constructs again.

TABLE 3A

TABLE 3B

The results listed in Table 3A show that all CFDV DNA fragments according to the invention also in bacteri are active as a promoter and have a higher activity than the CaMV 35S promoter (see construct pRT 35S). Across from the construct pRT CF4, which promotor a CFDV DNA fragment contains the repeated structures (RPT), the 52 bp Sequence, the TATAA sequence and the stem-loop structure in the Be nucleotides 941-971, but does not contain any DNA Portions of the open reading frames ORF1, ORF2 and also ORF3 ent holds, shows the construct pRT 35S with the CaMV 35S promoter only less than 10% of its activity.

Claims (10)

1. CFDV virus DNA fragment, this is the stem-loop structure, not the one or the other Translation start (s) for the ORF1 open reading frame and / or ORF2 includes. 2. CFDV virus DNA fragment according to claim 1, characterized in that it comprises the repeated RPT structures, the 52 bp sequence and the TATAA sequence in addition to the "stem-loop" structure includes. 3. CFDV virus DNA fragment according to claim 1, characterized, it is nucleotides 211 to 991, 409 to 991, 611 to 991 or 711 to 991, wherein for numbering the Nucleotides the 5'-end of the cleavage of the circular CFDV DNA with the restriction endonuclease XhoI resulting linearized DNA has been assigned the position 1. 4. DNA fragment, that of one of the CFDV virus DNA fragments after one of the Claims 1 to 3 by substitution, deletion, insertion or Modification of single nucleotides or smaller groups is derived from nucleotides and a comparable one Having promoter activity as the starting fragment. 5. Use of one or more DNA fragments after one of claims 1 to 4 as a promoter.   6. Use of one or more DNA fragments according to claim 5 as a promoter in bacteria or yeasts. 7. Use of one or more DNA fragments according to claim 5 as a promoter for tissue-specific expression of genes in transgenic plants. 8. Use of one or more DNA fragments according to claim 7 for the phloem-specific expression of genes in transgenic Plants. 9. Use of one or more DNA fragments after one of claims 1 to 4 for the preparation of chimeric Constructs for transient and stable expression. 10. Transgenic plants, plant parts, transformed Plant, yeast or bacterial cells using A DNA according to any one of claims 1 to 4 are.