IE83968B1 - Transit peptide DNA sequence - Google Patents
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- IE83968B1 IE83968B1 IE1999/0458A IE990458A IE83968B1 IE 83968 B1 IE83968 B1 IE 83968B1 IE 1999/0458 A IE1999/0458 A IE 1999/0458A IE 990458 A IE990458 A IE 990458A IE 83968 B1 IE83968 B1 IE 83968B1
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Description
PATENTS ACT, 1992 99/0458 TRANSIT PEPTIDE DNA SEQUENCE BAYER CROPSCIENCE SA The present invention relates to novel transit peptide DNA sequences, to novel chimeric genes and to their use in plants for conferring on them an increased tolerance to herbicides of the phosphonomethylglycine family. It also relates to the plant cells transformed of these genes, by means to the transformed plants regenerated from these cells as well as to the plants derived from crossbreedings using these transformed plants.
Glyphosate, sulfosate or fosametine are broad- spectrum systemic herbicides of the phosphonomethyl— glycine family. They act essentially as competitive inhibitors of 5—(enolpyruvyl)shikimate-3—phosphate synthase (EC 2.5.1.19) or EPSPS in relation to PEP (phosphoenolpyruvate). After their application to the plant, they are translocated inside the plant where they accumulate in the rapidly growing parts, in particular the caulinary and root apexes, causing the deterioration and even the destruction of sensitive plants.
Plastidial EPSPS, the main of the target of these products, is an enzyme aromatic amino acid biosynthesis pathway which is encoded by one or more nuclear genes and synthesized. in the form of a cyto- plasmic precursor and then imported into the plastids where it accumulates in its mature form.
The tolerance of plants to glyphosate and to products of the family is obtained by the stable intro- duction inside their genome of an EPSPS gene of plant or bacterial origin mutant or nonmutant with respect to the characteristics of the inhibition of the product of this gene by glyphosate. Given the mode of action of glypho- sate and the degree of tolerance to glyphosate of the product of the genes used, it is useful to be able to express the product of translation of this gene so as to permit its substantial accumulation in plastids.
It is known, 4,535,060, to of the N—(phosphonomethyl)glycine or glyphosate, by introducing for example fronu American Patent confer on a plant a tolerance to a herbicide abovementioned type, in particular into the plant genome a gene encoding an EPSPS carrying at least one mutation making this enzyme more resistant to its competitive inhibitor (glyphosate), after localization of the enzyme in the plastidial compartment (EP 0,189,707, EP 0,218,571 or W088/02402). these techniques need to be improved in order to achieve greater reliability in the use of these plants under agronomic conditions.
In the understood as meaning any differentiated. multicellular organism capable of photosynthesis and "plant cell" any However, present description, "plant" is cell derived from a plant and capable of forming undif- ferentiated tissues such as calluses or differentiated tissues such as embryos or plant sections, plants or seeds. subject of the present invention is the production of transformed plants increased having an tolerance to herbicides of the phosphonomethylglycine family by regenerating cells transformed. by means of novel chimeric genes comprising a gene for tolerance to these herbicides. The invention also relates to these novel chimeric genes, to the novel transit peptides which they contain as well as to the plants containing them which are made more tolerant by an accumulation of the mutant enzyme, in its mature form, in the plants.
More particularly, the subject of the invention is a chimeric gene for conferring on plants an increased target EPSPS, transcription, a tolerance to a herbicide having as its comprising, in the direction of promoter region, a transit peptide region, a sequence of a gene encoding a glyphosate tolerance enzyme and. an 3‘, region untranslated polyadenylation that the in the direction of transcription, signal region in characterized in transit peptide comprises, a transit peptide from a plant gene encoding a plastid-localized enzyme, a partial sequence of the N—terminal mature part of a plant gene encoding a plastid-localized enzyme, and then a second transit peptide from a plant gene encoding a plastid-localized enzyme.
The invention also relates to any DNA sequence of the transit peptide region defined above.
The transit peptides which can be used in the transit peptide region may be known per se and may be of plant origin, for example, derived from maize, The first and the second transit peptides may be identical, different. sunflower, peas, tobacco or the like. analogous or They may in addition each comprise one or more transit peptide units. Preferably, a sequence derived from the SSU of the ribulose 1,5—diphosphate carboxylase oxygenase (RuBisCo) gene is used.
The partial sequence of the N—terminal mature part is derived from a plant gene encoding a plastid- localized enzyme, such as for example a maize, sunflower or pea gene or the like, it being possible for the original plant species to be identical, analogous or different from that from which the first and second transit peptides are derived respectively. Furthermore, the partial sequence of the mature part may comprise a varying number of amino acids, generally from 10 to 40, preferably from 18 to 33. Preferably, a sequence derived from the SSU of the ribulose l,5—diphosphate carboxylase (RuBisCo) Construction of the entire transit region may be oxygenase gene is used. carried out in a manner known per se, in particular by The role of this to enable the release of a fusion or‘ any other suitable means. characteristic region is mature, native protein with a maximum efficiency. which may be used in the chimeric gene according to the coding sequence for herbicide tolerance invention encodes a mutant EPSPS having a degree of This particular by mutation of the EPSPS gene, glyphosate tolerance. obtained in sequence, may be of bacterial origin, for example derived from Salmonella typhymurium (and called in the text which follows "AroA gene"), or of plant origin, for example from petunia or from tomatoes. This sequence may comprise one or more lOl to Ser. alternatively the Gly 96 to Ala mutations. mutations, for example the Pro. mutation or The promoter region of the chimeric gene accor- ding to the invention may consist advantageously of at least one promoter of a which is gene expressed naturally in plants, that is to say promoters of viral origin such as that of 35S RNA of the cauliflower mosaic (CaMV35S) gene of a crop such as maize or sunflower. virus or of plant origin such as the small subunit of ribulose (RubisCO) The untranslated polyadenylation signal region l,5—diphosphate carboxylase in 3' of the chimeric gene according to the invention may be of any origin, for example bacterial, such as the nopaline synthase gene, or of plant origin, such as the small subunit of the maize or sunflower RubisCO.
The chimeric gene according to the invention may comprise, in addition to the above essential parts, an (linker) promoter region and the coding sequence which may be of untranslated intermediate region between the any origin, bacterial, viral or plant.
EXAMPLE 1: CONSTRUCTION OF A CHIMERIC GENE; The construction of the chimeric gene according to the invention is carried out using the following elements: ) "Double CaMV" of which has been duplicated): The CaMV35S promoter was isolated by Odell et_al. (1985). A pJO 5-2, containing 850 bp upstream of the site of initiation of transcription was with EcoRI—HindIII, the this fragment were made blunt using Klenow polymerase and the promoter (that is to say part clone, about cut ends of isolated fragment inserted at the Hincll site of the vector pUCl9 (Yannish—Perron et al., 1985). This promoter was digested with Clal, the ends filled using Klenow polymerase and then redigested with Hindlll. A HindIII— EcoRV fragment, isolated from the same initial promoter, was introduced between these two sites. The promoter thus obtained possesses a double amplification region upstream of the regulatory elements of the CaMV35S promoter. It was introduced in the form of a HindIII—EcoRI fragment into the vector pRPA—BL 150 A alpha 2, described in French /04130, cut with HindIII and EcoRI.
) Transit region: well as the mature protein elements used are derived Patent Application the two transit peptides as from the cloned cDNA of the small subunit of the gene of maize RubisCO whose gene has been described by Lebrun gt (1987), cloned CDNA of the subunit of the gene of sunflower RubisCO, (1987). called optimized transit peptide, al. and from the small isolated by Waksman et al.
More specifically, the transit region, comprises, in the direction of translation: — a transit peptide for the small subunit of sunflower RuBisCO, — an N—terminal sequence of 22 amino acids for the mature part of the small subunit of maize RuBisCO, - a transit peptide for the small subunit of maize RuBisCO.
The using this peptide is called pRPA—BL 410.
Other construct optimized transfer similar sequences which contain sequences of 10 to 40 and preferably 18 and 33 may be used amino acids respectively.
In order to provide a comparative element, another construction was carried out using a first transit peptide and the same nature sequence part but without a second transit peptide, (pRPA—BL 294).
) Structural gene: it is derived from the mutant gene at the position (Pro 101 to Ser) of EPSPS of Stalker et al. (provided by Calgene) according to the prior art Salmonella typhymurium (1985). The pMG34—2 linearized with XbaI and then treated with Vigna radiata the two blunt ends The clone obtained possesses an Ncol site a 17—bp Sall clone was isolated by clone was nuclease. After recutting with Smal, were ligated. in the site called well as This initiator ATG as downstream of the pRPA—BL 104.
) Polyadenylation signal region: stop codon. the fragment is derived from the nopaline synthase gene of pTi (Bevan et al., 1983). This site is contained in a 260-bp 1983; which was treated with Klenow polymerase Mbol fragment PCT 84/02913) and cloned in the SmaI site of M13 mp 18 in order to (Fraley et al., Patent Application introduce the BamHI and EcoRI sites at the 5' and 3 ends respectively.
After cutting with BamHI and treating with yigna radiata nuclease followed by cutting with EcoRI and treating with Klenow polymerase, the resulting fragment was introduced in the vector p—BL 20 (cf. French Patent Application 88/04130), cut by XbaI and BamHI and treated with Klenow’ polymerase. After recutting’ with SalI and Sstl, a fragment of about 0.4 kbp containing the 3' nos sequence on the side of the Sall site and the right end on the T—DNA side of the SstI site is obtained.
The assembly of the various elements was carried out in the following manner: "Transit peptide of the SSU of the maize RuBisCO/AroA gene" fusion: The transit peptide of the SSU of the maize RuBisCO gene is 192-bp EcoRI—SphI fragment obtained from the cDNA corresponding to the SSU gene of the maize RuBisCO gene, al. (1987), initiation derived from a described by Lebrun et NcoI the translation site possessing an site spanning Sphl for and an codon corresponding to transit cleavage site of peptide.
Translational fusion is the maize transit peptide and the bacterial EPSPS gene by obtained between treating the Sphl end with bacteriophage T4 polymerase and. by ligating it with the Klenow polymerase—treated Ncol end of the AroA gene from pRPA-BL lO4, EcoRI. recut with Transit the SSU of RuBisCO/sequence of 22 amino acids of the mature part of peptide of maize the SSU of maize RuBisCO/AroA gene fusion: a 228-bp EcoRI—HindII fragment of the cDNA of the SSU of the maize RubisCO gene is ligated with the Klenow polymerase—treated Ncol end of the AroA Similarly, pRPA—BL 104 and translational fusion is gene from recut with ECORI. A obtained between the transit peptide of the SSU of maize RuBisCO, the 22 amino acids of the mature part of the SSU of maize RuBisCO and the bacterial EPSPS gene.
Transit peptide of the SSU of sunflower RuBisCO: The fragment is derived from the CDNA isolated (1987). An SphI site of the transit peptide by Waksman and Freyssinet site was created at the cleavage according to the method of Zoller and Smith (1984. The transit peptide of the SSU of sunflower RuBisCO thus obtained is a 171-bp EcoRI—SphI fragment. the SSU of RuBisCO/sequence of 22 amino acids of the mature part of Transit peptide of sunflower the SSU of maize RuBisCO/AroA gene fusion: The construct containing the transit peptide of the SSU of maize RuBisCO/sequence of 22 amino acids of the SSU of maize RuBisCO of the mature part of the maize gene fusion was cut with 171-bp EcoRI—SphI corresponding to the transit peptide of the SSU of of the sunflower RubisCO gene. A resulting construct exhibits a substitu- tion of the EcoRI-Sphl fragments and is a translational the SSU of RuBisCO/sequence of 22 amino acids of the mature part of the SSU of maize RuBisCO/AroA gene. fusion "transit peptide of sunflower The EcoRI—SalI fragment was ligated with the Sall-Sstl fragment containing the 3' the right end of the T-DNA. nos sequence and The resulting EcoRI—SstI of the SSU of of the mature part of the SSU of maize RuBisCO/AroA gene/3' nos/T—DNA right end", is substituted for the EcoRI-Sstl fragment containing the right end of the T-DNA of the fragment, comprising "transit peptide sunflower RuBisCO/sequence of 22 amino acids plasmid 150 A alpha 2 containing the double CaMV promoter. The transcriptional fusion "double CaMV/transit peptide of the SSU of sunflower RuBisCO/sequence of 22 amino acids of the mature part of the SSU of maize RuBisCO/AroA gene/3' 150 A alpha 2 was called pRPA—BL 294. nos in the vector "Transit peptide of the SSU of sunflower RuBisCO/sequence of 22 amino acids of the SSU of maize RuBisCO/transit peptide of the SSU of maize RuBisCO/AroA gene" fusion: The above construct is cut with NcoI—HindIII, releasing the AroA gene. Next it is ligated with a .5 kbp NcoI-HindIII fragment containing the "transit peptide of the SSU of maize RuBisCO/AroA gene" fusion. A resulting construct exhibits a substitution of the Ncol-HindIII fragments and is a translational fusion "transit peptide of the SSU of sunflower RuBisCO/sequence of 22 amino acids of the SSU of the RuBisCO of the mature part of the maize gene/transit peptide of the SSU of maize RuBisCO/AroA gene".
The EcoRI-SalI fragment was ligated with the Sa1I—SstI fragment containing the 3' the right end of the T-DNA. nos sequence and The resulting EcoRI-Sstl of the SSU of sunflower RuBisCO/sequence of 22 amino acids of the SSU fragment comprising "transit peptide of the RuBisCO of the mature part of the maize gene/ transit peptide of the SSU of maize RuBisCO/AroA gene/3' nos/T—DNA right end" is substituted for the EcoRI—SstI fragment containing the right end of the T—DNA of the plasmid 150 A alpha 2 containing the double CaMV promo- The "double CaMV/transit peptide of the SSU of sunflower RuBisCO/sequence of 22 amino acids of the SSU of the RuBisCO of the mature part ter. transcriptional fusion of the maize gene/transit peptide of the SSU of maize RuBisCO/AroA gene/3' nos in the vector 150 A alpha 2 was called pRPA—BL 410.
EXAMPLE 2: RESISTANCE OF THE TRANSFORMED PLANTS . Transformation: The vector is introduced into the nononcogenic EHA 101 (Hood et al., 1987) carrying the cosmid pTVK 291 1986). The procedure of Agrobacterium strain (Komari et al., transformation method is the Horsh et al. (1985).
. Regeneration: based on The regeneration of the tobacco PBD6 SEITA France) Murashige and Skoog (source using foliar explants is carried out on a (MS) basic medium containing 30 g/l and 200 ug/ml of The explants are removed from greenhouse- or in vitro-grown of sucrose kanamycin. foliar plants method and transformed according to the foliar disc (Science 1985, Vol. 227, p. 1229-1231) successive stages: the first comprises the induction of on an MS medium supplemented with 30 g/l of 0.05 mg/l of of benzylaminopurine in three shoots sucrose (ANA) containing naphthylacetic acid and 2 mg/l (BAP), for 15 The stage are then developed by culturing on an MS medium supplemented with for 10 days. The developed shoots are then removed and they are days. shoots formed during this g/l of sucrose, but not containing hormone, cultured on an MS planting medium containing half the content of salts, vitamins and sugars and not containing hormone. After about 15 days, the deeply-rooted shoots are placed in soil.
. Measurement of the glyphosate tolerance: the weighing the mass of calluses extrapolated to 100 foliar a) In vitro: tolerance is measured by discs of 0.5 cm in diameter, an MS after 30 days of growth on with 30 g/l of 0.05 mg/l of naphthaleneacetic acid and 2 mg/l of BAP medium supplemented sucrose, containing 35 ppm of glyphosate and 200 micrograms/ml of kanamycin. Under these conditions, it is observed that for the tobacco plants modified by the chimeric gene of pRPA BL 410 according to the the mass of invention, calluses is 34 g whereas for the plants modified by the chimeric gene without a second transit peptide, the mass is only 12 g. b) In vivo: 30 plants derived from the regene- ration of the tobaccos transformed using pRPA—BL 294 and pRPA—BL 410 respectively are transferred to a greenhouse and treated at the 5-leaf stage by spraying with an aqueous suspension at a dose corresponding to 0.6 kg/ha After 21 days, examination is carried out of the plants relative to of glyphosate (Round up). a phenotypic _ 10 _ untransformed control plants. Under these conditions, it is observed that the plants transformed using pRPA—BL 410 the control plants are completely destroyed; the which differs from the preceding one by the absence of a possess a negligible phytotoxicity whereas moreover, plants transformed using a chimeric gene, second transit peptide, than 30% possess a phytotoxicity of not less destruction.
These results show the brought by the use of a chimeric gene according to the clearly improvement invention for the same gene encoding the glyphosate tolerance.
The the invention may be used as parents for producing lines and transformed plants according to hybrids having an increased tolerance to glyphosate.
Claims (26)
1. DNA sequence for a transit peptide, characterized in that it comprises, in the direction of transcription, a sequence encoding a first transit peptide from a plant gene encoding a plastid—localized enzyme, a partial sequence of the N—terminal mature part of a plant gene encoding a plastid—localized enzyme, and then a sequence encoding a second transit peptide from a plant gene encoding a plastid-localized enzyme.
2. DNA sequence according to Claim 1, characterized in that the sequence encoding the second transit peptide is derived from the same plant as the sequence encoding the first transit peptide.
3. DNA sequence according to Claim 1, characterized in that the sequence encoding the second transit peptide is derived from a plant which is different from that for the first transit peptide.
4. DNA sequence according to one of Claims 1 to 3, characterized in that at least one sequence encoding one of the transit peptides is derived from the small the (RuBisCO) gene. subunit of Ribulose—l,5-diphosphate carboxylase/oxygenase
5. DNA sequence according to one of Claims 1 to 4, characterized in that the sequence of the N—terminal nature part is obtained from the small subunit of the RuBisCO gene.
6. DNA sequence according to one of Claims 1 to 5, characterized in that at least one sequence encoding one of the transit peptides is derived from the same gene as the original one for the sequence of the mature part.
7. DNA sequence according to one of Claims 1 to 6, characterized in that at least one sequence encoding one of the transit peptides is derived from a maize gene.
8. DNA sequence according to one of Claims 1 to 7, characterized in that at least one sequence encoding one of the transit peptides is derived from a sunflower gene.
9. DNA sequence according to one of Claims 1 to 7, characterized in that the sequence of the mature part is derived from a maize gene.
10. DNA sequence according to one of Claims 1 to 7, characterized in that the sequence of the mature part is derived from a sunflower gene.
11. DNA sequence according to Claim 10, characterized in that the sequence of the nature part comprises from 10 to 40 amino acids.
12. DNA sequence according to one of Claims 1 to 11, characterized in that the sequence of the nature part comprises from 18 to 33 amino acids.
13. Chimeric gene for conferring on plants an increased tolerance to a herbicide having as its target the enzyme 5-(enolpyruvyl)shikimate—3—phosphate synthase (EPSPS), comprising, in the direction of transcription, a promoter region, a transit peptide region, a coding sequence for glyphosate tolerance and an untranslated polyadenylation signal region in 3', characterized in that the transit peptide region comprises a DNA sequence according to one of Claims 1 to 12.
14. Chimeric gene according to Clainx 13, characterized in that the coding sequence for glyphosate tolerance is of bacterial origin.
15. Chimeric gene according to Claim 13, characterized in that the coding sequence for glyphosate tolerance is of plant origin.
16. Process for constructing a chimeric gene accord- ing to one of Claims 13 to 15, characterized in that at least two transit peptide regions the and at least one sequence of suitable mature part of plastid— localized plant genes as well as at least one sequence encoding suitable glyphosate tolerance and a polyadenylation signal region are isolated respectively, and in that they are then assembled in the direction of transcription of the tolerance gene.
17. Vector for transforming plants, characterized in that it comprises a chimeric gene according to one of Claims 13 to 15.
18. Transformed plant cell, characterized in that it contains a chimeric gene according to one of Claims to l5.
19.l9. Transformed plant with improved tolerance to a herbicide having EPSPS as target, Characterized in that it was obtained from a cell according to Claim 18.
20. Plant according to Claim 19, characterized in that it is a dicotyledon.
21. Plant according to Clainl 19, characterized in that it is a monocotyledon.
22. Transformed plant with improved tolerance to a herbicide having EPSPS as target, characterized in that it is derived from crossing using the plants according to Claims 19 to 20.
23. Process for producing lines and hybrids having increased glyphosate tolerance, characterized in that the plants according to one of Claims 19 to 21 are used as parents.
24. Process for treating the plants according to one of Claims 19 to 22 or obtained by the process according to Claim 23, characterized in that a herbicide having EPSPS as target is applied to the plants.
25. Process according to Claim 24, characterized in that the herbicide is a broad-spectrum systemic herbicide of the phosphonomethylglycine family.
26. Process according to Claim 25, characterized in that the herbicide is glyphosate. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FRFRANCE05/03/19919102872 | |||
| FR9102872A FR2673643B1 (en) | 1991-03-05 | 1991-03-05 | TRANSIT PEPTIDE FOR THE INSERTION OF A FOREIGN GENE INTO A PLANT GENE AND PLANTS TRANSFORMED USING THIS PEPTIDE. |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| IE990458A1 IE990458A1 (en) | 2000-11-15 |
| IE83968B1 true IE83968B1 (en) | 2005-08-10 |
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