WO1994001565A1

WO1994001565A1 - Genes for altering plant metabolism

Info

Publication number: WO1994001565A1
Application number: PCT/GB1993/001385
Authority: WO
Inventors: Denis Joseph Murphy; David James Fairbairn; Stephen Peter Slocombe
Original assignee: The Minister Of Agriculture, Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland
Priority date: 1992-07-01
Filing date: 1993-07-01
Publication date: 1994-01-20
Also published as: AU4508393A; EP0603371A1; AU672613B2; GB9213969D0

Abstract

Genetic material in the form of DNA is provided for use in enabling plants to produce petroselinic acid by its incorporation into the plant's DNA. Previously uncharacterised enzymes derivable therefrom are also provided. Particularly provided are transgenic oilseed rape plants capable of production of petroselinic acid.

Description

GENES FOR ALTERING PLANT METABOLISM.

The present invention relates to genetic material in the form of DNA, its incorporation into plants such as to enable them to produce petroselinic acid, and to previously uncharacterised enzymes derivable therefrom.

The 1991 Brassica napus (rapeseed) harvest in the UK was a new record high yield at 1.3 million tons. This comes at a time of increased surpluses of many edible agricultural products, including seed oils. At the same time as the glut in edible oils, there is a world shortage of short to medium chain fatty acids, especially lauric acid (C₁₂) which has many industrial applications, e.g. as detergents. At present, lauric acid oils to the value of at least £120 million are imported from overseas in the form of palm oil or coconut oil. It is not possible to grow high lauric acid crops in the UK for climatic reasons. A more novel approach is to employ genetic engineering to produce a crop which yields an isomer of oleic acid, i.e. petroselinic acid. Unlike oleic acid, the latter can be refined by ozonolysis to give lauric acid and adipic acid which are useful for polymerisation to nylons and other high value polymers. There is a proven world wide commercial market in excess of 2 million tons (1987) for adipic acid. Therefore, petroselinic acid is the potential source of two industrial feedstocks for nylon and detergent manufacture.

Petroselinic acid is an unusual fatty acid which only occurs in some genera belonging to the order Umbelliflorae/Umbellales, eg. coriander (Coriandrum sativum) , carrot (Daucus carota) , and is a seed-specific fatty acid which accumulates in the storage oil to high levels, eg. 75% in coriander. The varieties of rapeseed currently grown in the UK are high in oleic acid (55~70- total oil) and low in erucic acid and glucosinoates. Petroselinic acid is not present in oilseed crops.

The enzyme which produces petroselinic acid has been named a delta₆ desaturase because it apparently introduces a double bond between the sixth and seventh carbons of a C_l8 fatty acid chain. There was no published information concerning this enzyme, it had not been purified and no protein or DNA sequence information was available up until the work of Cahoon et al was published, after the priority date of the present patent application (Proc. Nat. Acad. Sci. USA (1992) Vol 89- 11184-11188). These workers have speculated since that the enzyme may infact be a delta^desaturase which exerts its effect by acting on a palmityl-acyl carrier protein (l6:0-ACP); in that event it would be a palmitate desaturase rather than a stearoyl desaturase (Cahoon et al.(1993) Biotechnological Aspects of Plant Lipids: International Workshop, Carmona, Spain). In the present application this enzyme is termed 'petroselinic acid producing desaturase' (PAPD) .

Oleic acid is produced by a delta_gdesaturase (stearoyl acyl carrier protein delta_gdesaturase) and is a common fatty acid found in plant membranes and storage oils. The situation regarding the delta_g -desaturase is quite the reverse of the delta₆/_ήdesaturase/PAPD; 11 plant delta_gdesaturase cDNAs have been cloned and sequenced by several groups around the world in only two years. This is probably a reflection of the high homology at the protein and DNA levels between delta_gdesaturases from unrelated plants and the availability of antibody and DNA probes. Plant delta_gdesaturases are soluble enzymes, located in the plastid and require reduced ferrodoxin and molecular oxygen for activity.

With the advent of new plant genetic engineering methods it is now feasible to introduce new genes into crops such as rapeseed in order to create varieties capable of making new types of seed oil. By the introduction of a new gene into rapeseed it is possible to obtain a transgenic variety capable of high yields of petroselinic acid while maintaining other agronomically favourable characteristics such as low glucosinolate and erucic levels.

The present invention provides isolated cDNA sequences encoding for petroselinic acid producing desturase, 'PAPD' , as found in Coriandrum ffq .i iim. and further provides isolated cDNA sequences encoding for Brassica napus (rapeseed oil plant) delta_gdesaturase. The isolated PAPD cDNA encodes for the PAPD enzyme, which was not previously isolated or sequenced and thus enables production of the isolated enzyme and preparations enriched in that enzyme to be provided.

The present invention, having the aim of producing a transgenic rapeseed oil plant capable of expression of the PAPD enzyme enzyme present, eg. in Coriandrum sativum) . specifically provides the coriander PAPD and its cDNA, and using the cDNA or its complementary sequence as hybridisation probes or PCR primers, allows identification and isolation of PAPD from other plants using conventional recombinant DNA techniques. Furthermore, the present invention also provides antisense cDNA to this desaturase cDNA which may be used to down-regulate PAPD expression if so desired by incorporation into DNA of plants in the known manner.

The present invention further provides recombinant DNA constructs comprising the cDNA of the invention together with suitable regulatory sequences such as promotors and terminators. These regulatory sequences are preferably seed specific as that is the site of production of the fatty acid product realised in industrial processing of the plant product. Suitable known seed specific promotors are for example promotors of delta_gdesaturase, napin, cruciferin, oleosin and acyl carrier protein of Brassica napus. Suitable terminators are the NOS-3¹ (nopaline synthase) or OCS-3¹ (octopine synthase) terminators. These constructs have utility for incorporation into plant cell DNA to enable delta₆desaturase expression.

In order that the selection of suitable cloned cells containing constructs might be facilitated, marker sequences may also be incorporated such as Npt-II (for provision of kanamycin resistance) or Hpt (Hygromycin resistance) . Marker promotors may also be employed such as pnos (nopaline synthase) , pocs (octapine synthase) or 35s (caulimos, virus).

The present invention further provides isolated cDNA encoding the antisense sequence to the delta_gdesaturase of oilseed rape and its use to downregulate expression of delta_gdesaturase by incorporating it into the DNA of oilseed rape plants, particularly by incorporating it into the DNA of oilseed rape plant seeds. Such incorporation is carried out by producing a recombinant DNA construct which contains all or part of the cDNA in the antisense orientation together with appropriate regulatory sequences.

Suitable regulatory sequences are known in the art and have been described as used to regulate other delta_gdesaturase antisense cDNA expression (see Knutzon et al, Proc. Natl. Acad. Sci. USA Vol.89 pp2624-2628 (1992) . It will be realised by the person skilled in the art that known delta_gdesaturase cDNA may also be used to form antisense constructs for oilseed rape delta_gdesaturase down regulation, as is evidenced by the above Knutzon reference, and thus may be used in place of the delta_gdesaturase constructs of the present invention in down regulation in the transgenic oilseed rape plants that are ultimately to be provided and thus increase the amount of C_l8 fatty acid starting material for delta₆desaturase to convert to the petroselinic acid product, if that is indeed the route by which the final product is realised.

The present invention futher provides plants having PAPD expressing cDNA incorporated into their DNA and particularly provides transgenic Umbelliforae plants containing this cDNA, more particularly Brassica napus and rapus plants incorporating such material. The preferred plants comprise the desaturase constructs of the invention and more preferably also comprise the antisense delta_gdesaturase constructs of the invention or of the prior art. Such contructs may be incorporated into the plants DNA, preferably that of the seed tissue DNA, by known means, eg. by employing vectors such as the transformation mediator Agrobacterium (see Knutzon et al) .

The present invention, as stated above, provides petroselinic acid producing desaturase, PAPD, encoding cDNA and thus enables production of the isolated enzyme for the first time and thus provides for the raising of specific antibodies, polyclonal or monoclonal, by conventional techniques using the isolated enzyme as antigen. In order that specific antibodies to any such desaturase might be provided the present invention also provides a method of identification and isolation of such enzymes by use of the isolated coriander PAPD cDNA of the present invention, or its complementary sequence, as a probe to identify other related sequences by use of Southern (DNA) or Northern (RNA) blotting techniques from which the corresponding aminoacid sequence may be derived either schematically or physically by expression in organisms transformed by inclusion of the so identified sequences, eg. transformed E.coli.

In a further method of identification and isolation using the cDNA of the present invention it is possible to use sequences (eg degenerates) derived from that cDNA as polymerase chain reaction primers and to thus amplify the sequence of DNA native to other such plants than coriander to derive their particular sequences in a characteristic form. Necessarily that may entail some experimentation in determining parts of the coriander PAPD sequence that are conserved yet do not unduly result in amplification of delta_gdesaturase sequences. Suitable candidate sequences for amplification will be selectable by hybridisation with all or part of the coriander sequence, eg. by use of Northern blot analysis on RNA derived from plants actively producing petroselinic acid. Conditions for carrying out a Northern blot such that PAPDs may be distinguished from delta_gdesaturases are provided herein below in the Examples. The sequence SEQ ID No 1 provided below is a 1297 base sequence comprising the cDNA encoding for coriander PAPD together with that for an N-terminal transit peptide for plastidial localisation, with the amino acid sequence of the delta₆desaturase encoded by that nucleotide sequence provided below that. The open reading frame may be read from nucleotide bases encoding for the methionine at amino acid number ^~~ to that encoding for leucine at amino acid number 3^5 or may be read from the nucleotide bases encoding for methionine at amino acid number 14 . Since the priority date of this application, but before filing of the complete specification, Cahoon et al have reported (see above) that this transit sequence may be encoded by a DNA including an additional 5' sequence GCAAAAATGGCCATGA. The sequence given herein provides functionality in so far contains enough recognition features to allow targetting of the chloroplast.

Thus the present invention particularly provides cDNAs encoding for the desaturase amino acid sequence shown as SEQ ID No 2, amino acid sequence 14 to 381 or amino acid sequence 5 to 381 thereof; DNA having complementary sequence to them, or to antisense DNA derived from them; all in isolated form with the DNA also provided in vectored or cloned form. The DNAs may be labelled or may incorporate markers as is described above.

The present invention further particularly provides peptides comprising the full amino acid sequence SEQ ID No 2 and having petroselinic acid producing ability in plants, eg, coriandrum and brassica, or those consisting of amino acids 5 to 3^5 or 14 to 3^5 of that amino acid and fusion peptides comprising these sequences as may be produced by incorporation of their cDNA sequences into larger sequences encoding for other peptides as in known in the art.

The present invention also provides Brassica napus delta_gdesaturase cDNA and the enzyme itself, as described by or within SEQ ID No 3 and 4 respectively, in isolated, vectored or cloned form, unlabelled, labelled or marked as provided by conventional techniques.

The polynucleotide sequence of SEQ ID No 3 from 1221 to 1524 does not encode for the desaturase but is provided as an optional part of the isolated DNA. It will be realised that if downregulation of the delta_gdesaturase in the transgenic plants is to be achieved without similarly down-regulating the desired PAPD then only part or parts of the SEQ ID No 3 that do not target that DNA will be used in antisense form. An example of such a part is given and is designated SEQ ID No 5 to be used with sense strand SEQ ID No 6 .

EXAMPLE 1: PREPARATION OF cDNA ENCODING FOR CORIANDER DELTA_C/DELTA_GDESATURASE.

The cDNA of SEQ ID No 1 is prepared in facile manner from developing coriander seeds by use of RACE PCR (Rapid Amplification of C-DNA Ends, Polymerase Chain Reaction) . A cDNA library is prepared from seed stages high in petroselinic acid and synthetic primers are selected to target the SEQ ID No 1 and its complementary sequence in the known manner. The PCR reaction is carried out to provide desired 1297 basepair product. The duplex product may be used directly or may be cloned into a plasmid and then into a suitable host, eg. E. coli.

Suitable primers for specific amplification of the delta₆desaturase may be selected from any of the suitably sized sequences running from the ends of the 1297 base pair product as comparison of the PAPD and delta, sequences in these regions shows them to have a low homology and thus eg. 15 base length primers for the respective cDNA and complementary strands will be capable of specifically priming amplification of the required product.

The primers used for isolating DNA comprising the cDNA by RACE-PCR, (Frohman et al (1988) Proc. Nat. Acad. Sci. USA. Vol 85. 8998-9002) before it had been sequenced, were those designated SEQ ID No 7 and 8: SEQ ID No 7 5'" GGC TGC AGT GGA CIG CIG AxG AxG AA -3'

wherein at x the base is 0# A and 50 G in each position; this providing a degenerate oligonucleotide mixture; I is inosine; and the underlined bases form a PstI site.

The antisense strand primer used is the 3¹ RACE primer described by Frohman et al (1988):

SEQ ID No 8 5*- GAC TCG AGT CGA CAT CG -3'

It will be understood by those skilled in the art that efficient replication will be possible using directly complementary primers to the SEQ ID No 1 sequence and its complementary sequence.

EXAMPLE 2: DISTINGUISHING OF PAPD cDNA FROM DELTA_gDESATURASE cDNA.

It has been found that use of the following parameters with Northern blotting can identify delta₆desaturase RNA expression from E.coli clones transformed by vectors containing the aforesaid sequence; this blotting does not give hybridization with delta_gdesaturase RNA and thus provides a mechanism for the skilled man to determine when he has isolated the desired sequence.

Entire PAPD cDNA of 1297 base pairs (see SEQ ID No 1), derived by RACE-PCR of coriander seed DNA, was used as a probe in a form labelled by standard methods with ³²P.

The assay is a standard Northern Blot wherein RNA extract is prehybridized for 4 hours at 65°C then hybridized overnight (approx. 16 hours) at 65°C with labelled probe. The gel is washed and exposed to the film with low stringency wash and then washing is repeated with washes of higher stringency. The washes comprise the following compositions: Low stringency: (5 ^~~ SSPE) + 0.1J&SDS at room temperature. Medium stringency: (2 x SSPE) + 0.1J&SDS at 4θ°C. High stringency: (0.1 x SSPE) + O.ltfSDS at 65°C. 20 x SSPE is 3.6M NaCl: 0.2M Sodium Phosphate: 0.02M EDTA and has pH 7.7.

EXAMPLE : PRODUCTION OF CONSTRUCTS. The pUC vector derivative SLJ4D4 (see Jones et al, (1992) Transgenic Res. 1, 285-297) was provided and and manipulated using standard techniques (Sambrooke et al, (1989) Molecular cloning: a laboratory manual Section edition, Cold Spring Harbor Laboratory Press. New York). The CaMV35S promoter sequence was removed using EcoRl/Ncol digestion and replaced by the delta_gdesaturase promoter. The GUS sequence was removed by Ncol/BamHl digestion and replaced by either the PAPD or the antisense delta_gdesaturase cDNA. Once the various combinations of promoter and cDNA sequences had been assembled in SLJ4D4, the EcoRl/Hind3 fragment containing them was subcloned into the EcoRl/Hind3 sites of the binary vector (see Jones et al ibid for details of vector SLJ44026) . The PAPD has an internal Hind3 site and therefore required an EcoRl and partial Hind3 digest for cloning into the binary vector.

Antisense-delta_gdesaturase/promoter construct: The 5' end of the B. napus delta_gdesaturase was amplified by PCR. The antisense primer had an Ncol site and the sense primer had a BamHl site, at the 5' ends respectively. The PCR product (SEQ ID No 5 and 6) was digested with Ncol/BamHl and cloned into Bluescript. Bluescript primers were used to sequence the cloned PCR product and check for any errors introduced during the amplification step. Once the sequence of the PCR product had been confirmed it was removed by digestion with Ncol/BamHl and cloned into the Ncol/BamHl sites of SLJ4D4 and delta₉ desaturase/GUS plasmids. Constructs for antisense use are those with low homology to the PAPD sequence whereby effect on PAPD production is minimized.

Constructs as described are inserted into Agrobacterium as described by Knutzon et al and this vector used to transfer them to seed of the target Brassica plants by conventional methods. SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT:

(A NAME: THE MINISTER OF AGRICULTURE FISHERIES AND FOOD (B STREET: WHITEHALL PLACE (C CITY: LONDON (E COUNTRY: UNITED KINGDOM (F POSTAL CODE (ZIP) : SW1A 2HH

(A NAME: DENIS JOSEPH MURPHY (B STREET: 81 CHRISTCHURCH ROAD (C CITY: NORWICH (D STATE: NORFOLK (E COUNTRY: UNITED KINGDOM (F POSTAL CODE (ZIP): NR2 3NG

(A NAME: DAVID JAMES FAIRBAIRN (B STREET: 14 JUBILEE TERRACE (C CITY: NORWICH (D STATE: NORFOLK (E COUNTRY: UNITED KINGDOM (F POSTAL CODE (ZIP): NR72HG

(A NAME: STEPHEN PETER SLOCOMBE (B STREET: 33 EADE ROAD (C CITY: NORWICH (D STATE: NORFOLK (E COUNTRY: UNITED KINGDOM (F POSTAL CODE (ZIP): NR3 3EH

(ϋ) TITLE OF INVENTION: GENES FOR ALTERING PLANT METABOLISM

(iϋ) NUMBER OF SEQUENCES: 8

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release £1.0, Version £1.25 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: GB 9213969.0

(B) FILING DATE: Ol-JUL-1992

(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1297 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: CORIANDRUM SATIVUM (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 3.-1145

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

AA CTG AAT GCC CTC ATG ACT CTT CAG TGC CCA AAA AGG AAC ATG TTT 7 Leu Asn Ala Leu Met Thr Leu Gin Cys Pro Lys Arg Asn Met Phe 1 5 10 15

ACG AGA ATT GCC CCT CCT CAA GCA GGG AGA GTG AGA TCA AAG GTG TCC 95 Thr Arg He Ala Pro Pro Gin Ala Gly Arg Val Arg Ser Lys Val Ser 20 25 30

ATG GCT TCA ACT CTT CAT GCT AGC CCA CTG GTG TTC GAC AAG CTG AAG 143 Met Ala Ser Thr Leu His Ala Ser Pro Leu Val Phe Asp Lys Leu Lys 35 40

GCT GGG AGG CCT GAG GTG GAT GAA TTG TTC AAC TCT CTG GAG GGT TGG 191 Ala Gly Arg Pro Glu Val Asp Glu Leu Phe Asn Ser Leu Glu Gly Trp 50 55 60

GCC AGG GAC AAC ATC CTT GTG CAC CTG AAA TCC GTA GAG AAC TCA TGG 239 Ala Arg Asp Asn He Leu Val His Leu Lys Ser Val Glu Asn Ser Trp 65 70 75

CAG CCG CAA GAC TAT CTG CCC GAT CCC ACA TCC GAT GCA TTT GAA GAT 287 Gin Pro Gin Asp Tyr Leu Pro Asp Pro Thr Ser Asp Ala Phe Glu Asp 80 85 90 95

CAA GTC AAG GAG ATG AGA GAA CGG GCC AAG GAC ATC CCT GAT GAA TAC 335 Gin Val Lys Glu Met Arg Glu Arg Ala Lys Asp He Pro Asp Glu Tyr 100 105 HO

TTT GTT GTT CTT GTT GGA GAC ATG ATC ACT GAA GAG GCA CTC CCA ACT 383 Phe Val Val Leu Val Gly Asp Met He Thr Glu Glu Ala Leu Pro Thr 115 120 125

TAC ATG TCT ATG CTT AAC AGA TGT GAT GGC ATT AAG GAT GAC ACT GGC 431 Tyr Met Ser Met Leu Asn Arg Cys Asp Gly He Lys Asp Asp Thr Gly 130 135 140

GCT CAA CCT ACT TCT TGG GCC ACT TGG ACC AGG GCT TGG ACT GCT GAG 479 Ala Gin Pro Thr Ser Trp Ala Thr Trp Thr Arg Ala Trp Thr Ala Glu 145 150 155

GAG AAC CGC CAT GGC GAT CTT CTC AAC AAG TAT CTT TAT CTC TCT GGC 527 Glu Asn Arg His Gly Asp Leu Leu Asn Lys Tyr Leu Tyr Leu Ser Gly 160 165 170 175

CGA GTT GAT ATG AGG ATG ATT GAG AAG ACT ATT CAA TAT CTT ATC GGC 575 Arg Val Asp Met Arg Met He Glu Lys Thr He Gin Tyr Leu He Gly 180 185 190 GTCAGATCT GGTTATGTAA AACTTATATT TAAATGAATG TTGGATTTAA TA 1297

1265

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3δl amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Leu Asn Ala Leu Met Thr Leu Gin Cys Pro Lys Arg Asn Met Phe Thr 1 5 10 15

Arg He Ala Pro Pro Gin Ala Gly Arg Val Arg Ser Lys Val Ser Met 20 25 30

Ala Ser Thr Leu His Ala Ser Pro Leu Val Phe Asp Lys Leu Lys Ala 35 40 45

Gly Arg Pro Glu Val Asp Glu Leu Phe Asn Ser Leu Glu Gly Trp Ala 50 55 60

Arg Asp Asn He Leu Val His Leu Lys Ser Val Glu Asn Ser Trp Gin 65 70 75 80

Pro Gin Asp Tyr Leu Pro Asp Pro Thr Ser Asp Ala Phe Glu Asp Gin 85 90 95

Val Lys Glu Met Arg Glu Arg Ala Lys Asp He Pro Asp Glu Tyr Phe 100 105 HO

Val Val Leu Val Gly Asp Met He Thr Glu Glu Ala Leu Pro Thr Tyr 115 120 125

Met Ser Met Leu Asn Arg Cys Asp Gly He Lys Asp Asp Thr Gly Ala 130 135 140

Gin Pro Thr Ser Trp Ala Thr Trp Thr Arg Ala Trp Thr Ala Glu Glu 145 150 155 160

Asn Arg His Gly Asp Leu Leu Asn Lys Tyr Leu Tyr Leu Ser Gly Arg 165 170 175

Val Asp Met Arg Met He Glu Lys Thr He Gin Tyr Leu He Gly Ser 180 185 190

Gly Met Asp Thr Lys Thr Glu Asn Cys Pro Tyr Met Gly Phe He Tyr 195 200 205

Thr Ser Phe Gin Glu Arg Ala Thr Phe He Ser His Ala Asn Thr Ala 210 215 220

Lys Leu Ala Gin His Tyr Gly Asp Lys Asn Leu Ala Gin Val Cys Gly 225 230 235 240 Asn He Ala Ser Asp Glu Lys Arg His Ala Thr Ala Tyr Thr Lys He 245 250 255

Val Glu Lys Leu Ala Glu He Asp Pro Asp Thr Thr Val He Ala Phe 260 265 270

Ser Asp Met Met Arg Lys Lys He Gin Met Pro Ala His Ala Met Tyr 275 280 285

Asp Gly Ser Asp Asp Met Leu Phe Lys His Phe Thr Ala Val Ala Gin 290 295 300

Gin He Gly Val Tyr Ser Ala Trp Asp Tyr Cys Asp He He Asp Phe

310 315 320

Leu Val Asp Lys Trp Asn Val Ala Lys Met Thr Gly Leu Ser Gly Glu 325 330 335

Gly Arg Lys Ala Gin Glu Tyr Val Cys Ser Leu Ala Ala Lys He Arg 340 345 350

Arg Val Glu Glu Lys Val Gin Gly Lys Glu Lys Lys Ala Val Leu Pro 355 360 365

Val Ala Phe Ser Trp He Phe Asn Arg Gin He He He 370 375 380

(2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1524 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: BRASSICA NAPUS

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 27..1223

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

CTGAAAGAAC GTCAAACCTT CAAAAA ATG GCA TTG AAG TTT AAT CCT TTG GTA 53

Met Ala Leu Lys Phe Asn Pro Leu Val 1 5

TCT CAG CCA TAC AAA CTC GCT TCC TCG GCT CGT CCG CCA GTC TCT ACT 101 Ser Gin Pro Tyr Lys Leu Ala Ser Ser Ala Arg Pro Pro Val Ser Thr 10 15 20 25 TTC AGA TCT CCC AAG TTC CTA TGC CTC GCT TCT TCT TCT TCT CCT GCT 149 Phe Arg Ser Pro Lys Phe Leu Cys Leu Ala Ser Ser Ser Ser Pro Ala 30 35 40

CTC AGC TCC AAG GAG GTC GAG AGC TTG AAG AAG CCA TTC ACC CCA CCA 197 Leu Ser Ser Lys Glu Val Glu Ser Leu Lys Lys Pro Phe Thr Pro Pro 45 50 55

AGG GAA GTC CAC CTT CAA GTC CTG CAC TCC ATG CCA CCC CAA AAG ATC 245 Arg Glu Val His Leu Gin Val Leu His Ser Met Pro Pro Gin Lys He 60 65 70

GAA ATC TTC AAA TCC ATG GAA GAC CGG GCC GAG CAG AAC CTT CTA CCT 293 Glu He Phe Lys Ser Met Glu Asp Arg Ala Glu Gin Asn Leu Leu Pro 75 80 85

CAC CTC AAA GAC GTG GAG AAG TCA TGG CAG CCC CAG GAC TTC TTA CCC 34l His Leu Lys Asp Val Glu Lys Ser Trp Gin Pro Gin Asp Phe Leu Pro 90 95 100 105

GAC CCT GCT TCC GAC GGT TTC GAA GAT CAG GTA AAA GAG TTA AGA GAA 389 Asp Pro Ala Ser Asp Gly Phe Glu Asp Gin Val Lys Glu Leu Arg Glu 110 115 120

AGA GCA AGA GAG CTC CCA GAT GAT TAC TTC GTT GTC TTG GTT GGA GAC 437 Arg Ala Arg Glu Leu Pro Asp Asp Tyr Phe Val Val Leu Val Gly Asp 125 130 135

ATG ATC ACA GAA GAA GCG CTT CCC ACC TAC CAA ACA ATG TTA AAC ACT 485 Met He Thr Glu Glu Ala Leu Pro Thr Tyr Gin Thr Met Leu Asn Thr 140 145 150

TTG GAT GGA GTA AGG GAT GAG ACT GGC GCT AGC CCC ACT TCA TGG GCC 533 Leu Asp Gly Val Arg Asp Glu Thr Gly Ala Ser Pro Thr Ser Trp Ala 155 160 165

GTT TGG ACT AGA GCT TGG ACC GCT GAA GAG AAT CGC CAC GGT GAT CTT 58I Val Trp Thr Arg Ala Trp Thr Ala Glu Glu Asn Arg His Gly Asp Leu 170 175 180 185

CTC AAT AAG TAT CTT TAC TTG TCT GGT CGT GTT GAC ATG AGG CAG ATT 629 Leu Asn Lys Tyr Leu Tyr Leu Ser Gly Arg Val Asp Met Arg Gin He 190 195 200

GAA AAG ACT ATT CAG TAC CTG ATT GGT TCC GGA ATG GAT CCA CGC ACA 677 Glu Lys Thr He Gin Tyr Leu He Gly Ser Gly Met Asp Pro Arg Thr 205 210 215

GAG AAC AAC CCT TAC CTT GGC TTC ATC TAC ACT TCA TTC CAA GAG AGA 725 Glu Asn Asn Pro Tyr Leu Gly Phe He Tyr Thr Ser Phe Gin Glu Arg 220 225 230

GCC ACC TTC GTC TCT CAC GGC AAC ACA GCT CGC CAA GCC AAA GAG CAC 773 Ala Thr Phe Val Ser His Gly Asn Thr Ala Arg Gin Ala Lys Glu His 235 240 245

GGA GAC CTC AAG CTA GCG CAA ATC TGC GGG ACA ATA GCT GCA GAC GAG 821 Gly Asp Leu Lys Leu Ala Gin He Cys_Gly Thr He Ala Ala Asp Glu 250 255 260 265 AAG CGT CAC GAG ACA GCT TAC ACC AAG ATA GTT GAG AAG CTT CTC GAG 869 Lys Arg His Glu Thr Ala Tyr Thr Lys He Val Glu Lys Leu Leu Glu 270 275 280

ATT GAT CCT GAC GGC ACT GTG GTG GCC TTT GCG GAT ATG ATG AGG AAG 917 He Asp Pro Asp Gly Thr Val Val Ala Phe Ala Asp Met Met Arg Lys 285 90 295

AAA ATC TCG ATG CCT GCT CAC TTG ATG TAC GAT GGG CGT GAT GAT AAC 965 Lys He Ser Met Pro Ala His Leu Met Tyr Asp Gly Arg Asp Asp Asn 300 305 310

CTC TTT GAC AAC TTC TCC TCC GTG GCT CAG AGG CTC GGT GTT TAC ACT 1013 Leu Phe Asp Asn Phe Ser Ser Val Ala Gin Arg Leu Gly Val Tyr Thr 315 320 325

GCC AAA GAC TAT GCG GAC ATT CTT GAG TTT TTG GCC GGG AGG TGG AGG 1061 Ala Lys Asp Tyr Ala Asp He Leu Glu Phe Leu Ala Gly Arg Trp Arg 330 335 340 345

ATC GAG AGC TTG ACT GGG CTT TCG GGT GAA GGG AAC AAA GCG CAA GAG 1109 He Glu Ser Leu Thr Gly Leu Ser Gly Glu Gly Asn Lys Ala Gin Glu 350 355 360

TAT TTG TGT GGG TTG ACT CCG AGA ATC AGG AGG TTG GAT GAG AGA GCT 1157 Tyr Leu Cys Gly Leu Thr Pro Arg He Arg Arg Leu Asp Glu Arg Ala 365 370 375

CAA GCA AGA GCC AAG AAA GGA CCT AAG ATT CCT TTT AGC TGG ATA CAT 1205 Gin Ala Arg Ala Lys Lys Gly Pro Lys He Pro Phe Ser Trp He His 380 385 390

GAC AGA GAA GTG CAG CTC TGAACACAAA GGACAAAAGA CATAATAAAA 1253

Asp Arg Glu Val Gin Leu 395

CCATTTTCTC TCTCTCTCTC TCCGTTCATC ATTTGATATG TCTGCTCTTG AAGTTGGTGT 1313

AGATTACTAT GGTTTCCTGA TAATGTTCGT TGGTCTAGTT ACAAAGTTGA GAAGCAGTGT 1373

CTTAGTAACT TTGTGTTTTT CTTTCAGTGT CTTATGTTTG GTCTTTTAGT AAACTTCTGG 1433

TAGTTAAAAA CAGTTGAGCG TTTGGGTCTG TACTCAGTTT TCACTGTGGA GTTTTGTTCT 1493

AGTTGAAGTT AGTTTTTGTG TGTTAAAAAA A 1524

(2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 399 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Ala Leu Lys Phe Asn Pro Leu Val Ser Gin Pro Tyr Lys Leu Ala

1 10 15 Ser Ser Ala Arg Pro Pro Val Ser Thr Phe Arg Ser Pro Lys Phe Leu 20 25 30

Cys Leu Ala Ser Ser Ser Ser Pro Ala Leu Ser Ser Lys Glu Val Glu 35 40 45

Ser Leu Lys Lys Pro Phe Thr Pro Pro Arg Glu Val His Leu Gin Val 50 55 60

Leu His Ser Met Pro Pro Gin Lys He Glu He Phe Lys Ser Met Glu 65 70 75 80

Asp Arg Ala Glu Gin Asn Leu Leu Pro His Leu Lys Asp Val Glu Lys 85 90 95

Ser Trp Gin Pro Gin Asp Phe Leu Pro Asp Pro Ala Ser Asp Gly Phe 100 105 HO

Glu Asp Gin Val Lys Glu Leu Arg Glu Arg Ala Arg Glu Leu Pro Asp 115 120 125

Asp Tyr Phe Val Val Leu Val Gly Asp Met He Thr Glu Glu Ala Leu 130 135 140

Pro Thr Tyr Gin Thr Met Leu Asn Thr Leu Asp Gly Val Arg Asp Glu 145 150 155 160

Thr Gly Ala Ser Pro Thr Ser Trp Ala Val Trp Thr Arg Ala Trp Thr 165 170 175

Ala Glu Glu Asn Arg His Gly Asp Leu Leu Asn Lys Tyr Leu Tyr Leu 180 185 190

Ser Gly Arg Val Asp Met Arg Gin He Glu Lys Thr He Gin Tyr Leu 195 200 205

He Gly Ser Gly Met Asp Pro Arg Thr Glu Asn Asn Pro Tyr Leu Gly 210 215 220

Phe He Tyr Thr Ser Phe Gin Glu Arg Ala Thr Phe Val Ser His Gly 225 230 235 240

Asn Thr Ala Arg Gin Ala Lys Glu His Gly Asp Leu Lys Leu Ala Gin 245 250 255

He Cys Gly Thr He Ala Ala Asp Glu Lys Arg His Glu Thr Ala Tyr 260 265 270

Thr Lys He Val Glu Lys Leu Leu Glu He Asp Pro Asp Gly Thr Val 275 280 285

Val Ala Phe Ala Asp Met Met Arg Lys Lys He Ser Met Pro Ala His 290 295 300

Leu Met Tyr Asp Gly Arg Asp Asp Asn Leu Phe Asp Asn Phe Ser Ser 305 310 315 320

Val Ala Gin Arg Leu Gly Val Tyr Thr Ala Lys Asp Tyr Ala Asp He 325 330 335 Leu Glu Phe Leu Ala Gly Arg Trp Arg He Glu Ser Leu Thr Gly Leu 340 345 350

Ser Gly Glu Gly Asn Lys Ala Gin Glu Tyr Leu Cys Gly Leu Thr Pro 355 360 365

Arg He Arg Arg Leu Asp Glu Arg Ala Gin Ala Arg Ala Lys Lys Gly 370 375 380

Pro Lys He Pro Phe Ser Trp He His Asp Arg Glu Val Gin Leu 385 390 395

(2) INFORMATION FOR SEQ ID NO: :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 264 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: YES

(vi) ORIGINAL SOURCE:

(A) ORGANISM: BRASSICA NAPUS

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

CCATGGATTT GAAGATTTCG ATCTTTTGGG GTGGCATGGA GTGCAGGACT TGAAGGTGGA 60

CTTCCCTTGG TGGGGTGAAT GGCTTCTTCA AGCTCTCGAC CTCCTTGGAG CTGAGAGCAG 120

GAGAAGAAGA AGAAGCGAGG CATAGGAACT TGGGAGATCT GAAAGTAGAG ACTGGCGGAC l8θ

GAGCCGAGGA AGCGAGTTTG TATGGCTGAG ATACCAAAGG ATTAAACTTC AATGCCATTT 240

TTTGAAGGTT TGACGTTCTT TCAG 264

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 264 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: BRASSICA NAPUS (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CTGAAAGAAC GTCAAACCTT CAAAAAATGG CATTGAAGTT TAATCCTTTG GTATCTCAGC 60 CATACAAACT CGCTTCCTCG GCTCGTCCGC CAGTCTCTAC TTTCAGATCT CCCAAGTTCC 120 TATGCCTCGC TTCTTCTTCT TCTCCTGCTC TCAGCTCCAA GGAGGTCGAG AGCTTGAAGA 180 AGCCATTCAC CCCACCAAGG GAAGTCCACC TTCAAGTCCT GCACTCCATG CCACCCCAAA 240 AGATCGAAAT CTTCAAATCC ATGG 264

(2) INFORMATION FOR SEQ ID NO: 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL (iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: CORIANDRUM SATIVUM

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GGCTGCAGTG GACNGCNGAR GARAA 24 2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: CORIANDRUM SATIVUM

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

GACTCGAGTC GACATCG 17

Claims

CLAIMS,

1. Isolated, recombinant or cloned DNA encoding for an enzyme responsible for introducing the olefinic bond present at position 6 of petroselinic acid when produced by tissue of plants of group Umbelliforae, or antisense DNA thereto.

2. DNA or as claimed in claim 1 in labelled or marker containing form.

3- DNA as claimed in claim 3 wherein the enzyme has the amino acid sequence of that enzyme when obtained from a plant of genus Coriandrum or Daucus.

4. DNA as claimed in claim 4 wherein the enzyme has the amino acid sequence of that enzyme when obtained from a plant of species Coriandrum sativum or Daucus carota.

5- DNA as claimed in any one of claims 1 to 5 and having the sequence SEQ ID No 1 or the sequence between positions 14 to 1145 or 4l to 1145 thereof.

6. DNA as claimed in claim 1 and being obtainable by carrying out a PCR reaction using Coriandrum sativum seed genomic DNA as template and primers targeted at the ends of the sequence and its respective complementary sequence respectively.

7. Isolated or enriched protein that is capable of introducing the olefinic bond present in petroselinic acid produced in plant tissue, as derived by expression using a DNA as claimed in any one of claims 1 to 7-

8. A protein as claimed in claim 8, wherein the plant tissue is that of a plant of genus Coriandrum.

9- A protein as claimed in claim 9 being that of Coriandrum sativum. 10. A protein as claimed in any one of claims 7 to 9 produced from cDNA as claimed in any one of claims 1 to 7-

11. A recombinant DNA construct comprising cDNA as claimed in any one of claims 1 to 7 together with one or more regulatory DNA sequences capable of regulating expression of the enzyme for which it encodes.

12. A recombinant DNA construct as claimed in claim 11 comprising a regulatory sequence that is a promotor for transcription of the cDNA.

13. A recombinant DNA construct as claimed in claim 12 comprising a regulatory sequence that is a terminator for transcription of the cDNA.

14. A recombinant DNA construct as claimed in any one of claims 11 to 13 wherein the regulatory sequences are seed specific.

15- A vector comprising a recombinant DNA construct as claimed in any one of claims 11 to 14.

16. A transgenic plant comprising DNA incorporating a construct or vector as claimed in any one of claims 11 to 15.

17. A transgenic plant as claimed in claim 16 further comprising a delta_gdesaturase antisense DNA capable of down-regulating expression of the plants delta_gdesaturase.

19- A transgenic plant as claimed in claiml7 wherein the antisense DNA is incorporated into the DNA of its seeds.

20. A transgenic plant as claimed in any one of claims 16 to 19 wherein the plant is an Umbelliforae. 21. A transgenic plant as claimed in any one of claims 16 to 20 wherein the plant is a Brassica.

22. A transgenic plant as claimed in claim 21 wherein the plant is a Brassica napus or a Brassica rapus.

23. Seeds obtained from a transgenic plant as claimed in any one of claims 16 to 22.

24. Petroselinic acid obtained from a transgenic plant as claimed in any one of claims 16 to 22.

25. Petroselenic acid as claimed in claim 24 obtained from the seeds of transgenic plant.