Abstract:
A process for producing fatty acids or fatty acid derivatives from oleaginous plants. This process is characterized in that transgenic oleaginous plants are produced having on the one hand at least one gene coding for a lipase enzyme, the so-called lipase gene, and on the other hand, associated with this lipase gene, a promoter permitting an expression of the gene either in compartments different from the lipid accumulation compartments, or by exogenous induction. The seeds or fruits containing the plant lipids are collected, the seeds or fruits are crushed, if necessary after induction treatment, so as to bring the lipids and lipase into contact, the whole mixture is incubated in order to effect an enzymatic hydrolysis of the lipids, and the fatty acids or derivatives thereof are extracted.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is the 35 USC §371 National phase of International application PCT/FR95/00957 filed Jul. 18, 1995, which designated the United States of America. 
    
    
     FIELD OF THE INVENTION 
     The invention relates to a process for producing fatty acids or derivatives of fatty acids (esters or other derivatives) from oleaginous plants. The process of the invention applies in particular to oleoproteaginous plants such as rapeseed, sunflower, soya, kale, etc. The invention may in particular be used to produce biofuels (diesters), lubricants, phytosanitary adjuvants, detergents, etc., by converting the resultant fatty acids. 
     BACKGROUND OF THE INVENTION 
     Since 1970 numerous attempts have been made to replace products derived from petroleum (in particular fuels) by products obtained from vegetable matter so as to reduce the dependency on oil-producing countries and increase the markets and sales of agricultural products. The industrial route utilizing oleaginous plants as starting materials starts from the production of free fatty acids constituting the raw materials for the conversion industries and ends up with fuels, lubricants, etc. The lipids accumulated by the oleaginous plants may be converted into fatty acids by hydrolysis: two processes are currently used industrially to effect this conversion. 
     The first process consists in hydrolyzing the lipids after extraction by contacting the extracted lipids under conditions of heat and pressure with sulphuric acid/methanol or methanolic potassium hydroxide. Another process consists in carrying out the hydrolysis under similar conditions directly on the crushed seed material without prior extraction. Further details of these processes, which are the only ones used industrially to hydrolyze vegetable oils, may be found in the following reference: K. J. Harrington and C. d&#39;Arcy-Evans, Ind. Eng. Chem. Prod. Res. Dev. 1985, 24, 314-318. The well-known high operating costs, large industrial infrastructure, polluting nature of the effluents, production of glycerol as a by-product without an existing market. These processes are used on account of the lack of simple alternatives. 
     In addition, experiments have been carried out in the laboratory to effect the hydrolysis of lipids enzymatically by mixing a lipase with the lipids extracted from seeds (G. P. McNeill et al., JAOCS, Vol. 68 No. 1st January 1991, p. 1-5; S. M. Kim and J. S. Rhee, JAOCS Vo. 68 No. 7th July 1991, p. 499-503; C. Gancet, In Heterogeneous Catalysis And Fine Chemicals II 1991, Guisnet Editors, p. 93-104). However, these experiments have remained at the laboratory stage since the technique is incompatible with an industrial exploitation on account of the amounts of enzyme required and the cost of the latter. 
     It should be emphasized that processes are already known enabling enzymes to be produced from plants (patent WO-A-92/01042 and EP-A-0.449.376). These processes lead to the linexpensive production of enzymes which are then used, after or without having been isolated, in various industrial or food industry conversion processes. 
     Nevertheless, this teaching is completely unconnected with the problem in question, which concerns the production of fatty acids directly from oleaginous plants. 
     OBJECTS OF THE INVENTION 
     The present invention aims to provide a new solution to the problem of producing fatty acids from oleaginous plants. The invention seeks to provide a solution whose costs of implementation are considerably less than known processes (industrial chemical processes as well as laboratory enzymatic processes). 
     An object of the invention is thus to provide a process that can be carried out on an industrial scale under mild temperature and pressure conditions, which is simple to operate and is non-polluting, employs a small-scale infrastructure, and does not produce any harmful by-product. 
     Another object, associated with the previous one, is to enable the number of fatty acid production installations to be increased so that they can be sited close to the regions of cultivation of the oleaginous plants and thereby achieve savings in transportation of raw materials. 
     SUMMARY OF THE INVENTION 
     To this end, the process according to the invention for producing fatty acids or fatty acid derivatives from oleaginous plants is characterized in that: 
     oleaginous plants are produced 
     transgenic oleaginous plants are produced having, on the one hand, at least one gene coding for a lipase enzyme, the so-called lipase gene, and on the other hand, associated with this lipase gene, a promoter permitting an expression of the gene either in cellular, extracellular or tissue compartments other than those in which the plant lipids accumulate, or permitting exogenous induction, 
     the seeds or fruits containing the lipids of the plants are collected, 
     the seeds or fruits are crushed, if necessary after induction treatment, so as to bring into mutual contact the lipids and lipase contained in the seeds or fruits, p1 the mixture is allowed to incubate to effect an enzymatic hydrolysis of the lipids of the ground material under the catalytic action of the lipase contained in the said crushed material, 
     the fatty acids formed by the hydrolysis are extracted or are converted in order to obtain the desired fatty acid derivatives. 
     The process of the invention is thus an enzymatic hydrolysis process that enjoys the advantages of this type of process (mild operating conditions, absence of pollution, small-scale and inexpensive installations, no harmful by-products). In this process the plant itself is induced to produce the enzyme necessary for the subsequent transformation of the lipids, this enzyme being prevented from coming into contact prematurely with the lipids so as to avoid any risk of self-degradation of the plant before harvesting. The hydrolysis then takes place without the addition of exogenous enzyme by bringing the lipids and enzymes produced by the plant into contact. Such a process has a particularly low overall operating cost. The crushing and incubating units are light and small-scale and are known in the agricultural industry, which means that these operations can be carried out at the sites where the plants are harvested. 
     The production of the transgenic plants is carried out by first of all effecting the genetic transformation of a natural oleaginous plant, causing the genetically transformed plant to reproduce sexually so as to produce transgenic seeds, and then using these seeds to obtain transgenic plant progeny. 
     The initial genetic transformation consists, according to a presently well-known process, of generating an expression cassette containing the lipase gene and the expression promoter of this gene and introducing this expression cassette into the genome of the plant. 
     One of the essential characteristics of the process of the invention is that the promoter associated with the lipase gene is adapted to prevent any premature contact of the enzyme and lipids; this promoter may be of various types: it may either (1) direct the expression of the gene in compartments other than those where the lipids accumulate, or (2) initiate the expression of the gene at a suitable moment by exogenous induction. 
     In the first case, two types of expression cassettes may be used: 
     (1A) either an expression cassette comprising a lipase gene and a promoter controlling the expression of this gene in a cellular or tissue compartment different from the lipid accumulation compartment, 
     (1B) or an expression cassette comprising a constituent promoter and a lipase gene provided with an address sequence for cellular or extracellular compartments other than those where the lipids accumulate. 
     In the second case (2) the promoter used in the expression cassette is of a type that can be controlled in an exogenous manner by physical, chemical or biochemical signals, in particular is a stress promoter controlling the expression by applying a physical shock to the seeds or fruits. 
     By way of example, to produce fatty acids from oleoproteaginous plants, the operating procedure 1A outlined above may be employed: 
     the genetic transformation of the plant is carried out by producing an expression cassette comprising a lipase gene and a promoter controlling the expression of a specific protein of the seed, and introducing this expression cassette into the genome of the plant so as to express the lipase in the compartments of the seed where the specified protein accumulates, 
     the lipids and the lipase are brought into contact by simple crushing. 
     In the case of rapeseed, the promoter of the protein used is advantageously the napine promoter, which permits a large accumulation of lipase in the protein bodies of the seed, separated from the lipid globules. 
     The procedure 1B outlined above may be used regardless of the type of oleaginous plant, for example by choosing the constituent promoter 35S of CaMV (cauliflower mosaic virus) and the tobacco address sequence PR-S so as to direct the secretion of the produced lipases to the extracellular compartments. In this case too the lipids and lipases are brought into contact by simple crushing. 
     The procedure (2) outlined above may be used regardless of the type of oleaginous plant, for example by choosing the protease inhibition promoter isolated from potatoes, which controls gene expression in the case of injury or damage. The induction treatment that initiates the synthesis of the lipases may in this case be shelling or hulling of the seeds, carried out before crushing. 
     Regardless of the procedure that is chosen, preferably a gene is used coding for a non-specific lipase, i.e. characterized by a non-specific hydrolytic activity, in order to achieve a complete hydrolysis of the lipids accumulated by the plant and prevent interfering saponification reactions. However, for certain uses of fatty acids it is possible to cause the plant to produce lipases of specific hydrolytic activity so as to promote a certain type of hydrolysis (for example: monoacylglycerol lipase from penicyllium camembertii, effecting the hydrolysis of only one of the three fatty acid-glycerol bonds, so as to produce diacylglycerols). 
     In particular there may be used non-specific lipase genes, characterized by the following sequences or by sequences similar to the following sequences (the arrows define the coding part): 
     
         SEQUENCE I (SEQ ID NO: 1)    ▾1   GATGACAACT TGGTTGGTGG CATGACTTTG GACTTACCCA GCGATGCTCC51 TCCTATCAGC CTCTCTAGCT CTACCAACAG CGCCTCTGAT GGTGGTAAGG101   TTGTTGCTGC TACTACTGCT CAGATCCAAG AGTTCACCAA GTATGCTGGT151   ATCGCTGCCA CTGCCTACTG TCGTTCTGTT GTCCCTGGTA ACAAGTGGGA201   TTGTGTCCAA TGTCAAAAGT GGGTTCCTGA TGGCAAGATC ATCACTACCT251   TTACCTCCTT GCTTTCCGAT ACAAATGGTT ACGTCTTGAG AAGTGATAAA301   CAAAAGACCA TTTATCTTGT TTTCCGTGGT ACCAACTCCT TCAGAAGTGC351   CATCACTGAT ATCGTCTTCA ACTTTTCTGA CTACAAGCCT GTCAAGGGCG401   CCAAAGTTCA TGCTGGTTTC CTTTCCTCTT ATGAGCAAGT TGTCAATGAC451   TATTTCCCTG TCGTCCAAGA ACAATTGACC GCCCACCCTA CTTATAAGGT501   CATCGTTACC GGTCACTCAC TCGGTGGTGC ACAAGCTTTG CTTGCCGGTA551   TGGATCTCTA CCAACGTGAA CCAAGATTGT CTCCCAAGAA TTTGAGCATC601   TTCACTGTCG GTGGTCCTCG TGTTGGTAAC CCCACCTTTG CTTACTATGT651   TGAATCCACC GGTATCCCTT TCCAACGTAC CGTTCACAAG AGAGATATCG701   TTCCTCACGT TCCTCCTCAA TCCTTCGGAT TCCTTCATCC CGGTGTTGAA751   TCTTGGATGA AGTCTGGTAC TTCCAACGTT CAAATCTGTA CTTCTGAAAT801   TGAAACCAAG GATTGCAGTA ACTCTATCGT TCCTTTCACC TCTATCCTTG                                              {569851   ACCACTTGAG TTACTTTGAT ATCAACGAAG GAAGCTGTTT GTAAAACACT901   TGACGTGTTA CTCTAATTTT ATAATAAAAT TAAGTTTTTA TACAATSEQUENCE II (SEQ ID NO: 2)1  GTCGACCATT TCAGCCTGTT TTGCTCGCAA AACGACGCCG CGGGCGTGCG51 CTACCGCACA CTCCGTCGCT GGGCGTTGTG CGGGGAAGAT TCAAACGAGC101   GTTTCGCGCC GTAACAACCC GCTCTCTTCC GCTCTGCCAC GCAGGTTATG151   ACCGGCCGCC AGGAAGCCGC GGATTTCCTG GCCTGGAGGA AAAAAGCCGA201   AGCTGGCACG GTTCCTGGCG CAAGGGACAG CGAAGCGGTT CTCCCGGAAG251   GATTCGGGCG ATGGCTGGCA GGACGCGCCC CTCGGCCCCA TCAACCTGAG          ▾301   ATGAGAACAA CATGAAGAAG AAGTCTCTGC TCCCCCTCGG CCTGGCCATC351   GGTCTCGCCT CTCTCGCTGC CAGCCCTCTG ATCCAGGCCA GCACCTACAC401   CCAGACCAAA TACCCCATCG TGCTGGCCCA CGGCATGCTC GGCTTCGACA451   ACATCCTCGG GGTCGACTAC TGGTTCGGCA TTCCCAGCGC CTTGCGCCGT501   GACGGTGCCC AGGTCTACGT CACCGAAGTC AGCCAGTTGG ACACCTCGGA551   AGTCCGCGGC GAGCAGTTGC TGCAACAGGT GGAGGAAATC GTCGCCCTCA601   GCGGCCAGCC CAAGGTCAAC CTGATCGGCC ACAGCCACGG CGGGCCGACC651   ATCCGCTACG TCGCCGCCGT ACGTCCCGAC CTGATCGCTT CCGCCACCAG701   CGTCGGCGCC CCGCACAAGG GTTCGGACAC CGCCGACTTC CTGCGCCAGA751   TCCCACCGGG TTCGGCCGGC GAGGCAGTCC TCTCCGGGCT GGTCAACAGC801   CTCGGCGCGC TGATCAGCTT CCTTTCCAGC GGCAGCACCG GTACGCAGAA851   TTCACTGGGC TCGCTGGAGT CGCTGAACAG CGAGGGTGCC GCGCGCTTCA901   ACGCCAAGTA CCCGCAGGGC ATCCCCACCT CGGCCTGCGG CGAAGGCGCC951   TACAAGGTCA ACGGCGTGAG CTATTACTCC TGGAGCGGTT CCTCGCCGCT1001   GACCAACTTC CTCGATCCGA GCGACGCCTT CCTCGGCGCC TCGTCGCTGA1051   CCTTCAAGAA CGGCACCGCC AACGACGGCC TGGTCGGCAC CTGCAGTTCG1101   CACCTGGGCA TGGTGATCCG CGACAACTAC CGGATGAACC ACCTGGACGA1151   GGTGAACCAG GTCTTCGGCC TCACCAGCCT GTTCGAGACC AGCCCGGTCA                                                 ▾1201   GCGTCTACCG CCAGCACGCC AACCGCCTGA AGAACGCCAG CCTGTAGSEQUENCE III (SEQ ID NO: 3)1  GGGTGCATGC CAGCTCCCAC CGGACACCTG GCCCGTCGCT GAAACGTGTT                                          ▾51 TTCGCTTTCT CTACAAATCC AACAACAGAG AGGCACTACC ATGGGTATCT101   TTGACTATAA AAACCTTGGC ACCGAGGGTT CCAAAACGTT CTTCGCCGAT151   GCCATGGCGA TCACGTTGTA TTCCTATCAC AACCTGGATA ACGGCTTTGC201   CGTGGGCTAC CAGCACAACG GGTTGGGCTT GGGGCTACCG GCCACGCTGG251   TCGGTGCGCT GCTCGGCAGC ACGGATTCCC AGGGCGTGAT CCCTGGCATC301   CCGTGGAACC CGGATTCAGA AAAAGCCGCC CTTGAGGCGG TGCAGAAAGC351   CGGTTGGACA CCGATCAGCG CCAGTGCCCT GGGCTACGCC GGCAAGGTCG401   ATGCACGTGG CACCTTCTTT GGGGAAAAAG CCGGCTACAC CACGGCCCAG451   GTCGAGGTAC TCGGCAAATA CGATGACGCC GGCAAGCTGC TCGAAATCGG501   CATCGGTTTT CGTGGCACTT CGGGGCCACG GGAAACCTTG ATCAGCGACT551   CGATCGGCGA CTTGATCAGC GATCTGCTCG CGGCCCTGGG GCCCAAGGAT601   TACGCGAAAA ACTACGCCGG CGAAGCCTTC GGCGGCTTGC TCAAGAATGT651   TGCCGACTAC GCCGGTGCCC ATGGCCTGAC CGGCAAGGAC GTGGTGGTCA701   GCGGCCACAG CCTGGGCGGG CTGGCGGTCA ACAGCATGGC GGACTTGAGC751   AACTACAAAT GGGCGGGGTT CTACAAGGAC GCCAACTATG TTGCCTATGC801   CTCGCCGACC CAGAGTGCCG GCGACAAGGT GCTCAATATC GGTTACGAAA851   ACGACCCGGT GTTCCGCGCG CTGGACGGCT CGTCGTTTAA CCTGTCGTCG901   CTGGGCGTGC ACGACAAACC CCACGAGTCC ACCACCGATA ACATCGTCAG951   CTTCAACGAC CACTACGCCT CGACGCTGTG GAATGTGCTG CCGTTTTCCA1001   TCGTCAACCT GCCCACCTGG GTCTCGCATT TGCCGACGGC GTACGGCGAT1051   GGCATGACGC GCATCCTCGA GTCCGGCTTC TACGACCAGA TGACCCGTGA1101   CTCCACGGTG ATTGTTGCCA ACCTGTCCGA TCCGGCGCGG GCCAACACCT1151   GGGTGCAGGA CCTCAACCGC AATGCCGAGC CCCACAAGGG CAACACGTTC1201   ATCATCGGCA GCGACGGCAA CGACCTGATC CAGGGCGGCA ACGGTGCGGA1251   CTTTATCGAG GGTGGCAAAG GCAACGACAC GATCCGCGAC AACAGCGGGC1301   ACAACACCTT TTTGTTCAGC GGCCACTTTG GCAATGATCG CGTGATTGGC1351   TACCAGCCCA CCGACAAACT GGTGTTCAAG GACGTGCAAG GAAGCACCGA1401   CCTGCGTGAC CACGCGAAGG TGGTCGGCGC CGATACGGTG CTTACGTTTG1451   GGGCCGACTC GGTGACGCTG GTCGGCGTGG GGCATGGCGG GCTGTGGACG                     ▾1501   GAGGGCGTGG TGATCGGCTG ATTACTCACG CAACCGATCA GTGCCAGTGC1551   TGCCCCCGCC AGCCACCGCC CCAATTGGGC CGGTGGGGGT AGCCATAGCCSEQUENCE IV (SEQ ID NO: 4)1  GGGCGATGGC TGGCAGGACG CGCCCCTCGG CCCCATCAAC CTGAGATGAG51 AACAACATGA AGAAGAAGTC TCTGCTCCCC CTCGGCCTGG CCATCGGCCT                                   ▾101   CGCCTCTCTC GCTGCCAGCC CTCTGATCCA GGCCAGCACC TACACCCAGA151   CCAAATACCC CATCGTGCTG GCCCACGGCA TGCTCGGCTT CGACAATATC201   CTCGGGGTCG ACTACTGGTT CGGCATTCCC AGCGCCTTGC GCCGTGACGG251   TGCCCAGGTC TACGTCACCG AAGTCAGCCA GTTGGACACC TCGGAAGTCC301   GCGGCGAGCA GTTGCTGCAA CAGGTGGAGG AAATCGTCGC CCTCAGCGGC351   CAGCCCAAGG TCAACCTGAT CGGCCACAGC CACGGCGGGC CGACCATCCG401   CTACGTCGCC GCCGTACGTC CCGACCTGAT GCCTTCCGCC ACCAGCGTCG451   GCGCCCCGCA CAAGGGTTCG GACACCGCCG ACTTCCTGCG CCAGATCCCA501   CCGGGTTCGG CCGGCGAGGC AGTCCTCTCC GGGCTGGTCA ACAGCCTCGG551   CGCGCTGATC AGCTTCCTTT CCAGCGGCAG CGCCGGTACG CAGAATTCAC601   TGGGCTCGCT GGAGTCGCTG AACAGCGAGG GGGCCGCGCG CTTCAACGCC651   AAGTACCCGC AGGGCATCCC CACCTCGGCC TGCGGCGAAG GCGCCTACAA701   GGTCAACGGC GTGAGCTATT ACTCCTGGAG CGGTTCCTCG CCGCTGACCA751   ACTTCCTCGA TCCGAGCGAC GCCTTCCTCG GCGCCTCGTC GCTGACCTTC801   AAGAACGGCA CCGCCAACGA CGGCCTGGTC GGCACCTGCA GTTCGCACCT851   GGGCATGGTG ATCCGCGACA ACTACCGGAT GAACCACCTG GACGAGGTGA901   ACCAGGTCTT CGGCCTCACC AGCCTGTTCG AGACCAGCCC GGTCAGCGTC                                        ▾951   TACCGCCAGC ACGCCAACCG CCTGAAGAAC GCCAGCCTGT AGGACCCCGG1001   CCGGGGCCTC GGCCCCGGCC CTTTCCCGGA AGCCCCCTCG CGTGAAGAAA1051   ATCCTCCTGC TGATTCCACT GGCGTTCGCC GCCAGCCTGG CCTGGTTCGTSEQUENCE V (SEQ ID NO: 5)   ▾1  CAGGCCCCCA CGGCCGTTCT TAATGGCAAC GAGGTCATCT CTGGTGTCCT51 TGGGGCAAG GTTGATACCT TTAAGGGAAT TCCATTTGCT GACCCTCCTG101   TTGGTGACTT GCGGTTCAAG CACCCCCAGC CTTTCACTGG ATCCTACCAG151   GGTCTTAAGG CCAACGACTT CAGCTCTGCT TGTATGCAGC TTGATCCTGG201   CAATGCCATT TCTTGGCTTG ACAAAGTCGT GGGCTTGGGA AAGATTCTTC251   CTGATAACCT TAGAGGCCCT CTTTATGACA TGGCCCAGGG TAGTGTCTCC301   ATGAATGAGG ACTGTCTCTA CCTTAACGTT TTCCGCCCTG CTGGCACCAA351   GCCTGATGCT AAGCTCCCCG TCATGGTTTG GATTTACGGT GGTGCCTTTG401   TGTTTGGTTC TTCTGCTTCT TACCCTGGTA ACGGCTACGT CAAGGAGAGT451   GTGGAAATGG GCCAGCCTGT TGTGTTTGTT TCCATCAACT ACCGTACCGG501   CCCCTATGGA TTCCTGGGTG GTGATGCCAT CACCGCTGAG GGTAACACCA551   ACGCTGGTCT GCACGACCAG CGCAAGGGTC TCGAGTGGGT TAGCGACAAC601   ATTGCCAACT TTGGTGGTGA TCCCGACAAG GTCATGATTT TCGGTGAGTC651   CGCTGGTGCC ATGAGTGTTG CTCACCAGCT TGTTGCCTAC GGTGGTGACA701   ACACCTACAA CGGAAAGAAG CTTTTCCACT CTGCCATTCT TCAGTCTGGC751   GGTCCTCTTC CTTACTTTGA CTCTACTTCT GTTGGTCCCG AGAGTGCCTA801   CAGCAGATTT GCTCAGTATG CCGGATGTGA TGCCAGCGCC AGTGACAATG851   AAACTCTGGC TTGTCTCCGC AGCAAGTCCA GCGATGTCTT GCACAGTGCC901   CAGAACTCGT ACGATCTCAA GGACCTGTTT GGCCTGCTCC CTCAATTCCT951   TGGATTTGGT CCCAGACCCG ACGGCAACAT TATTCCCGAT GCCGCTTATG1001   AGCTCTACCG CAGCGGTAGA TACGCCAAGG TTCCCTACAT TACTGGTAAC1051   CAGGAGGATG AGGGTACTAT TCTTGCCCCC GTTGCTATTA ATGCTACCAC1101   GACTCCCCAT GTTAAGAAGT GGTTGAAGTA CATTTGTAGC GAGGCTTCTG1151   ACGCTTCGCT TGATCGTGTT TTGTCGCTCT ACCCCGGCTC TTGGTCGGAG1201   GGTGCGCCAT TCCGCACTGG TATTCTTAAT GCTCTGACCC CTCAGTTCAA1251   GCGCATTGCT GCCATTTTCA CTGATTTGCT GTTCCAGTCT CCTCGTCGTG1301   TTATGCTTAA CGCTACCAAG GACGTCAACC GCTGGACTTA CCTTGCCACC1351   CAGCTCCATA ACCTCGTTCC ATTTTTGGGT ACTTTCCATG GTAGTGATCT1401   TCTTTTCCAA TACTACGTGG ACCTTGGCCC ATCTTCTGCT TACCGCCGCT1451   ACTTTATCTC GTTTGCCAAC CACCACGACC CCAACGTTGG CACCAACCTG1501   AAACAGTGGG ATATGTACAC TGATGCAGGC AAGGAGATGC TTCAGATTCA1551   TATGGTTGGT AACTCTATGA GAACTGACGA CTTTAGAATC GAGGGAATCT                                    ▾1601   CGAACTTTGA GTCTGACGTT ACTCTCTTCG GTTAASEQUENCE VI (SEQ ID NO: 6)1                                                 ▾1  ATGGAGCTCG CTCTTGCGCT CCTGCTCATT GCCTCGGTGG CTGCTGCCCC51 CACCGCCACG CTCGCCAACG GCGACACCAT CACCGGTCTC AACGCCATCA101   TCAACGAGGC GTTCCTCGGC ATTCCCTTTG CCGAGCCGCC GGTGGGCAAC151   CTCCGCTTCA AGGACCCCGT GCCGTACTCC GGCTCGCTCG ATGGCCAGAA201   GTTCACGCTG TACGGCCCGC TGTGCATGCA GCAGAACCCC GAGGGCACCT251   ACGAGGAGAA CCTCCCCAAG GCAGCGCTCG ACTTGGTGAT GCAGTCCAAG301   GTGTTTGAGG CGGTGCTGCC GCTGAGCGAG GACTGTCTCA CCATCAACGT351   GGTGCGGCCG CCGGGCACCA AGGCGGGTGC CAACCTCCCG GTGATGCTCT401   GGATCTTTGG CGGCGGGTTT GAGGTGGGTG GCACCAGCAC CTTCCCTCCC451   GCCCAGATGA TCACCAAGAG CATTGCCATG GGCAAGCCCA TCATCCACGT501   GAGCGTCAAC TACCGCGTGT CGTCGTGGGG GTTCTTGGCT GGCGACGAGA551   TCAAGGCCGA GGGCAGTGCC AACGCCGGTT TGAAGGACCA GCGCTTGGGC601   ATGCAGTGGG TGGCGGACAA CATTGCGGCG TTTGGCGGCG ACCCGACCAA651   GGTGACCATC TTTGGCGAGC TGGCGGGCAG CATGTCGGTC ATGTGCCACA701   TTCTCTGGAA CGACGGCGAC AACACGTACA AGGGCAAGCC GCTCTTCCGC751   GCGGGCATCA TGCAGCTGGG GGCCATGGTG CCGCTGGACG CCGTGGACGG801   CATCTACGGC AACGAGATCT TTGACCTCTT GGCGTCGAAC GCGGGCTGCG851   GCAGCGCCAG CGACAAGCTT GCGTGCTTGC GCGGTGTGCT GAGCGACACG901   TTGGAGGACG CCACCAACAA CACCCCTGGG TTCTTGGCGT ACTCCTCGTT951   GCGGTTGCTG TACCTCCCCC GGCCCGACGG CGTGAACATC ACCGACGACA1001   TGTACGCCTT GGTGCGCGAG GGCAAGTATG CCAACATCCC TGTGATCATC1051   GGCGACCAGA ACGACGAGGG CACCTTCTTT GGCACCCTGC TGTTGAACGT1101   GACCACGGAT GCCCAGGCCC GCGAGTACTT CAAGCAGCTG TTTGTCCACG1151   CCAGCGACGC GGAGATCGAC ACGTTGATGA CGGCGTACCC CGGCGACATC1201   ACCCAGGGCC TGCCGTTCGA CACGGGTATT CTCAACGCCC TCACCCCGCA1251   GTTCAAGAGA ATCCTGGCGG TGCTCGGCGA CCTTGGCTTT ACGCTTGCTC1301   GTCGCTACTT CCTCAACCAC TACACCGGCG GCACCAAGTA CTCATTCCTC1351   CTGAAGCAGC TCCTGGGCTT GCCGGTGCTC GGAACGTTCC ACTCCAACGA1401   CATTGTCTTC CAGGACTACT TGTTGGGCAG CGGCTCGCTC ATCTACAACA1451   ACGCGTTCAT TGCGTTTGCC ACGGACTTGG ACCCCAACAC CGCGGGGTTG1501   TTGGTGAAGT GGCCCGAGTA CACCAGCAGC CTGCAGCTGG GCAACAACTT1551   GATGATGATC AACGCCTTGG GCTTGTACAC CGGCAAGGAC AACTTCCGCA                                                 ▾1601   CCGCCGGCTA CGACGCGTTG TTCTCCAACC CGCCGCTGTT CTTTGTGTAA 
    
     The identification of the six aforementioned sequences is as follows in the &#34;EMBL&#34; and &#34;GENE BANK&#34; databank: 
     SEQUENCE I (SEQ ID NO:1) 
     LOCUS:RCHLIPASE, 946 bp ss mRNA PLN 23/01/93 
     DEFINITION:Rhizopus niveus mRNA for lipase, partial sequence 
     ACCESSION:D 12 680 
     SEQUENCE II (SEQ ID NO:2) 
     LOCUS:PALIPAG 1247 bp DNA BCT 21/07/93 
     DEFINITION:P airuginosa lip A gene for lipase 
     ACCESSION:X 63 390 S 43 732 
     SEQUENCE III (SEQ ID NO:3) 
     LOCUS:PSELIPASEE, 1700 bp ds-DNA BCT 15/04/92 
     DEFINITION:Pseudomonas fluorescens lipase gene, coplete cds 
     ACCESSION:M.86 350 
     SEQUENCE IV (SEQ ID NO:4) 
     LOCUS:PSELIPL, 1282 bp ds-DNA BCT 17/04/92 
     DEFINNITION:Pseudomonas sp. Lipl gene for lipase 
     ACCESSION:D 10166 D 90398 
     SEQUENCE V (SEQ ID NO:5) 
     LOCUS:GCU 02387, 1635 bp DNA PLN 07/10/93 
     DEFINITION:Geotrichum candidum NRRLY-553 Lipase gene, partial eds 
     ACCESSION:UO 2367 
     SEQUENCE VI (SEQ ID NO:6) 
     LOCUS:CCLIP1, 1733 bp DNA PLN 27/04/93 
     DEFINITION:C. Cylindracea LIP1 gene for lipase 
     ACCESSION:X 64703 
     The sequence I (SEQ ID NO:1) corresponds to a cDNA of Rhizopus niveus, the sequence II (SEQ ID NO:2) may be isolated from the genome of Pseudomonas aeruginosa, sequence III (SEQ ID NO:3) from Pseudomonas fluorescens, sequence IV (SEQ ID NO:4) from Pseudomonas sp, sequence V (SEQ ID NO:5) from Geotricum candidum, and sequence VI (SEQ ID NO:6) from Candida cylindracea. 
     In addition, the introduction of the expression cassette: lipase gene/expression promoter of this gene, into the genome of the oleaginous plant may be effected by any known protocol. 
     For example, according to the most common protocol at the present time, this expression cassette may be introduced into the genome of somatic cells of the plant by a transfer by means of the bacterium Agrobacterium tumefaciens. This introduction into the somatic cells of the plant may also be effected by any other known technique, in particular by electroporation, biolistics or by microinjection. 
     It is also possible to introduce the expression cassette into the genome of microspores of the plant by electroporation or biolistics. 
     In the protocol provided later by way of example, the technique of electroporation is described for introducing the cassettte into the microspores of rapeseed. 
     Reference may be made to the following document &#34;P. J. J. Hooykaas and R. A. Schilperoort, TIBS August 1985, p. 305-309&#34; for more details on the transfer technique by means of the bacterium Agrobacterium tumefaciens. It will be recalled that this technique consists in introducing the relevant expression cassette into the Ti plasmid of the bacterium, in particular by thermal shock, followed by contact between the bacterium and the leaf discs of the plant, allowing the whole to incubate until the expression cassette is transferred into the genome of the cells of the foliar discs, and cultivating these foliar discs on a succession of media to regenerate the transgenic plants. 
     Reference may also be made to the following document &#34;J. A. Russell et al., In Vitro Cell. Dev. Biol., 1992, 28P, p. 97-105&#34; for more details of the biolistics technique. It will be recalled that this technique consists in attaching the plasmid containing the expression cassette to gold or tungsten microspheres, shooting these microspheres by means of a particle gun onto the cells of the plant to be transformed, and cultivating these cells until regeneration of the transgenic plants has occurred. 
     Reference may be made to the following document &#34;Crossway A and A1, 1986, Mol. Gen. Genet 202, 179-185&#34; for more details of the technique of microinjection. It will be recalled that this technique consists in injecting the plasmid containing the expression cassette into protoplasts or very young embryos by means of microsyringes, and cultivating the protoplasts until regeneration of the transgenic plants has occurred. 
     After the lipase and lipids have been brought into contact by crushing, the mixture is left to incubate so as to effect enzymatic hydrolysis. This incubation is carried out under normal conditions, in particular between 20° C. and 60° C., for the time required to achieve a total or almost total hydrolysis. 
     The fatty acids produced by the hydrolysis are extracted and then worked up by any known process, in particular by liquid/liquid extraction using a non-polar solvent such as chloroform or hexane. 
     According to another procedure, it is possible to carry out an in situ conversion of the fatty acids to obtain fatty acid derivatives, which are then extracted. For example the fatty acids produced by the hydrolysis may be methylated in situ by mixing them with methanol under acid catalysis and ultrasound so as to convert the acids into methyl esters, which are then extracted by a liquid/liquid extraction using a non-polar solvent. 
     The present application provides, as a novel product, any oleaginous plant or oleaginous plant seed of a variety that is not protectable by a plant breeding certificate, which includes in its genome an expression cassette containing at least one gene coding for a lipase enzyme, associated with a promoter permitting an expression of the gene in cellular, extracellular or tissue compartments different from the lipid compartments of the plant or seed. 
     The promoter associated with the lipase gene may be a specific cellular expression or tissue expression promoter. This promoter may also be a constituent promoter, in which case the lipase gene is provided with an address sequence for cellular or extracellular compartments different from the lipid accumulation compartments. 
     The present application also covers any oleaginous plant or oleaginous plant seed of a variety that is not protectable by a plant breeding certificate, which includes in its genome an expression cassette having at least one gene coding for a lipase enzyme, associated with a promoter permitting an expression of the said gene by exogenous induction, in particular by a stress. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The following description together with the accompanying diagram provides by way of example a protocol for implementing the process of the invention; FIG. 1 of the diagram illustrates the preparation of the genetic material to be transferred. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     1. PROTOCOL FOR OBTAINING TRANSGENIC RAPESEED PLANTS EXPRESSING A LIPASE GENE 
     a) Plant material 
     Rapeseed (Brassica napus, Var Tapidor) seeds are used, which are commercially available. 
     The seeds are sown in a greenhouse and cultivated under normal conditions. The state of health of the plants is strictly monitored. 
     The young buds (size less than 3.5 mm) are picked, sterilized in sodium hypochlorite for 30 minutes, and the microspores are extracted from the anthers by crushing in a Waring Blender in the medium of Huang et al (Huang et al. 1990, Plant. Cell. Rep. 8, 594-597). After filtering through a 5 μm mesh size metal sieve, the microspores are recovered by centrifugation for 5 minutes at 100 xg (protocol described in Jardinaud et al., 1993, Plant. Sci. 93, 177-184). 
     b) Genetic structure 
     The genetic structure that is adopted utilizes the napine promoter, the cDNA of Rhizopus niveus lipase and the NOS terminator. The napine promoter governs the expression of this protein in a protein compartment of the seed different from that in which the lipids accumulate. The whole arrangement is introduced into the PRT1 plasmid containing the pat selection gene used to screen the transformants so as to form the pRTlL structure. 
     Details of the structure are shown in FIG. 1 of the drawing. 
     The napine promoter designated &#34;Prom. Nap&#34; was isolated by Professor Rask&#39;s team (Stalberg K. et al, 1993, Plant Molecular Biology, 23:671-683). The Rhizopus niveus lipase cDNA, designated &#34;Lip&#34;, was isolated by the Central Research Institute (Kugimiya et al., 1992, Biosci. Biotech. Biochem. 56, 716-719). An initiation codon, designated &#34;start codon&#34;, compatible with the 5&#39; end of the cDNA (available on the market) is grafted onto this end. A terminator designated NOS extracted from the pRT1 plasmid mentioned hereinbelow is grafted onto the 3&#39; end, designated &#34;lip&#34;, of the resultant arrangement, and the Nap promoter is grafted onto the 5&#39; end. 
     The expression cassette thus obtained is introduced into a plasmid designated &#34;Blue-Script&#34; (commercial name) so that it can be amplified. 
     The amplified expression cassette is then extracted from &#34;Blue-Script&#34; and introduced into a plasmid designated pRT1, which was prepared by grafting the promoter designated CaMV35S onto the 5&#39; end of the &#34;pat&#34; gene coding for phosphinothricine acetyl transferase (selection gene), and the NOS terminator onto the 3&#39; end. 
     A plasmid designated pRTlL is obtained containing the desired expression cassette. 
     c) Gene transfer and production of transgenic plants 
     The microspores isolated in a) (10 6  microspores ml -1 ) are suspended in Brewbaker and Kwack&#39;s medium (J. L. Brewbaker and B. H. Kwack, 1963, Am. J. Bot. 50, p. 859-865) containing 13% sucrose and adjusted to pH 5.9. 50 μg per ml of the pRTlL plasmid are added to the medium and electrical pulses of 400 V/cm are applied for 10 ms to the suspension by means of a &#34;Jouan&#34; electroporation apparatus (trademark) (TRX, GHT) delivering square wave pulses. After 20 minutes&#39; rest the culture medium (Huang et al.) containing 100 mg/l of phosphinothricine is added to the microspores. The microspores are cultivated in the dark for 24 hrs at 35° C. and then at 25° C. After two weeks of cultivation an equal volume of new medium is added and the microspores are exposed to cycles of 16 hours of light per day followed by 8 hours of darkness. 
     After about 1 month the embryos are transferred to B 5  medium (Gamborg et al., 1979, Exp. Cell. Res. 50, 151-158) containing 1 ml/g of G3A 3  and 20 g/l of sucrose, to which 8 g/l of &#34;Bacto Agar&#34; (trademark) gelling agent is added. 
     The phosphinothricine-resistant regenerated plants are analyzed by Southern blotting so as to verify the presence in their genome of the sequence coding for lipase. The chromosome complement of the plants is doubled by colchicine (0.1 g/l plus a few drops of Teepol) and the resultant fertile diploid plant are self-fertilized. 
     The activity of the lipase is investigated in a limited number of seeds. The plants having seeds with the greatest lipase activity are retained and the seeds are used to propagate the plants until enough seeds are obtained to perform the hydrolysis experiments on the seed lipids with endogenous lipase. 
     2. ENZYMATIC HYDROLYSIS OF THE LIPIDS OF THE RESULTANT RAPESEED SEEDS 
     The seeds are crushed and the crushed material is placed in an incubator kept at a constant temperature of 40°. The crushed material is constantly agitated to increase the contact between the lipids and the lipase. 
     After 48 hours hydrolysis, the fatty acids are extracted with chloroform. The chloroform is evaporated and the fatty acids are recovered. 
     
         __________________________________________________________________________#             SEQUENCE LISTING- (1) GENERAL INFORMATION:-    (iii) NUMBER OF SEQUENCES: 6- (2) INFORMATION FOR SEQ ID NO:1:-      (i) SEQUENCE CHARACTERISTICS:#pairs    (A) LENGTH: 946 base     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (ii) MOLECULE TYPE: cDNA-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:- GATGACAACT TGGTTGGTGG CATGACTTTG GACTTACCCA GCGATGCTCC TC - #CTATCAGC  60- CTCTCTAGCT CTACCAACAG CGCCTCTGAT GGTGGTAAGG TTGTTGCTGC TA - #CTACTGCT 120- CAGATCCAAG AGTTCACCAA GTATGCTGGT ATCGCTGCCA CTGCCTACTG TC - #GTTCTGTT 180- GTCCCTGGTA ACAAGTGGGA TTGTGTCCAA TGTCAAAAGT GGGTTCCTGA TG - #GCAAGATC 240- ATCACTACCT TTACCTCCTT GCTTTCCGAT ACAAATGGTT ACGTCTTGAG AA - #GTGATAAA 300- CAAAAGACCA TTTATCTTGT TTTCCGTGGT ACCAACTCCT TCAGAAGTGC CA - #TCACTGAT 360- ATCGTCTTCA ACTTTTCTGA CTACAAGCCT GTCAAGGGCG CCAAAGTTCA TG - #CTGGTTTC 420- CTTTCCTCTT ATGAGCAAGT TGTCAATGAC TATTTCCCTG TCGTCCAAGA AC - #AATTGACC 480- GCCCACCCTA CTTATAAGGT CATCGTTACC GGTCACTCAC TCGGTGGTGC AC - #AAGCTTTG 540- CTTGCCGGTA TGGATCTCTA CCAACGTGAA CCAAGATTGT CTCCCAAGAA TT - #TGAGCATC 600- TTCACTGTCG GTGGTCCTCG TGTTGGTAAC CCCACCTTTG CTTACTATGT TG - #AATCCACC 660- GGTATCCCTT TCCAACGTAC CGTTCACAAG AGAGATATCG TTCCTCACGT TC - #CTCCTCAA 720- TCCTTCGGAT TCCTTCATCC CGGTGTTGAA TCTTGGATGA AGTCTGGTAC TT - #CCAACGTT 780- CAAATCTGTA CTTCTGAAAT TGAAACCAAG GATTGCAGTA ACTCTATCGT TC - #CTTTCACC 840- TCTATCCTTG ACCACTTGAG TTACTTTGAT ATCAACGAAG GAAGCTGTTT GT - #AAAACACT 900#                946TTT ATAATAAAAT TAAGTTTTTA TACAAT- (2) INFORMATION FOR SEQ ID NO:2:-      (i) SEQUENCE CHARACTERISTICS:#pairs    (A) LENGTH: 1257 base     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (ii) MOLECULE TYPE: cDNA-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:- GTCGACCATT TCAGCCTGTT TTGCTCGCAA AACGACGCCG CGGGCGTGCG CT - #ACCGCACA  60- CTCCGTCGCT GGGCGTTGTG CGGGGAAGAT TCAAACGAGC GTTTCGCGCC GT - #AACAACCC 120- GCTCTCTTCC GCTCTGCCAC GCAGGTTATG ACCGGCCGCC AGGAAGCCGC GG - #ATTTCCTG 180- GCCTGGAGGA AAAAAGCCGA AGCTGGCACG GTTCCTGGCG CAAGGGACAG CG - #AAGCGGTT 240- CTCCCGGAAG GATTCGGGCG ATGGCTGGCA GGACGCGCCC CTCGGCCCCA TC - #AACCTGAG 300- ATGAGAACAA CATGAAGAAG AAGTCTCTGC TCCCCCTCGG CCTGGCCATC GG - #TCTCGCCT 360- CTCTCGCTGC CAGCCCTCTG ATCCAGGCCA GCACCTACAC CCAGACCAAA TA - #CCCCATCG 420- TGCTGGCCCA CGGCATGCTC GGCTTCGACA ACATCCTCGG GGTCGACTAC TG - #GTTCGGCA 480- TTCCCAGCGC CTTGCGCCGT GACGGTGCCC AGGTCTACGT CACCGAAGTC AG - #CCAGTTGG 540- ACACCTCGGA AGTCCGCGGC GAGCAGTTGC TGCAACAGGT GGAGGAAATC GT - #CGCCCTCA 600- GCGGCCAGCC CAAGGTCAAC CTGATCGGCC ACAGCCACGG CGGGCCGACC AT - #CCGCTACG 660- TCGCCGCCGT ACGTCCCGAC CTGATCGCTT CCGCCACCAG CGTCGGCGCC CC - #GCACAAGG 720- GTTCGGACAC CGCCGACTTC CTGCGCCAGA TCCCACCGGG TTCGGCCGGC GA - #GGCAGTCC 780- TCTCCGGGCT GGTCAACAGC CTCGGCGCGC TGATCAGCTT CCTTTCCAGC GG - #CAGCACCG 840- GTACGCAGAA TTCACTGGGC TCGCTGGAGT CGCTGAACAG CGAGGGTGCC GC - #GCGCTTCA 900- ACGCCAAGTA CCCGCAGGGC ATCCCCACCT CGGCCTGCGG CGAAGGCGCC TA - #CAAGGTCA 960- ACGGCGTGAG CTATTACTCC TGGAGCGGTT CCTCGCCGCT GACCAACTTC CT - #CGATCCGA1020- GCGACGCCTT CCTCGGCGCC CCTCGGCGCC TCGTCGCTGA CCTTCAAGAA CG - #GCACCGCC1080- AACGACGGCC TGGTCGGCAC CTGCAGTTCG CACCTGGGCA TGGTGATCCG CG - #ACAACTAC1140- CGGATGAACC ACCTGGACGA GGTGAACCAG GTCTTCGGCC TCACCAGCCT GT - #TCGAGACC1200- AGCCCGGTCA GCGTCTACCG CCAGCACGCC AACCGCCTGA AGAACGCCAG CC - #TGTAG1257- (2) INFORMATION FOR SEQ ID NO:3:-      (i) SEQUENCE CHARACTERISTICS:#pairs    (A) LENGTH: 1600 base     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (ii) MOLECULE TYPE: cDNA-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:- GGGTGCATGC CAGCTCCCAC CGGACACCTG GCCCGTCGCT GAAACGTGTT TT - #CGCTTTCT  60- CTACAAATCC AACAACAGAG AGGCACTACC ATGGGTATCT TTGACTATAA AA - #ACCTTGGC 120- ACCGAGGGTT CCAAAACGTT GTTCGCCGAT GCCATGGCGA TCACGTTGTA TT - #CCTATCAC 180- AACCTGGATA ACGGCTTTGC CGTGGGCTAC CAGCACAACG GGTTGGGCTT GG - #GGCTACCG 240- GCCACGCTGG TCGGTGCGCT GCTCGGCAGC ACGGATTCCC AGGGCGTGAT CC - #CTGGCATC 300- CCGTGGAACC CGGATTCAGA AAAAGCCGCC CTTGAGGCGG TGCAGAAAGC CG - #GTTGGACA 360- CCGATCAGCG CCAGTGCCCT GGGCTACGCC GGCAAGGTCG ATGCACGTGG CA - #CCTTCTTT 420- GGGGAAAAAG CCGGCTACAC CACGGCCCAG GTCGAGGTAC TCGGCAAATA CG - #ATGACGCC 480- GGCAAGCTGC TCGAAATCGG CATCGGTTTT CGTGGCACTT CGGGGCCACG GG - #AAACCTTG 540- ATCAGCGACT CGATCGGCGA CTTGATCAGC GATCTGCTCG CGGCCCTGGG GC - #CCAAGGAT 600- TACGCGAAAA ACTACGCCGG CGAAGCCTTC GGCGGCTTGC TCAAGAATGT TG - #CCGACTAC 660- GCCGGTGCCC ATGGCCTGAC CGGCAAGGAC GTGGTGGTCA GCGGCCACAG CC - #TGGGCGGG 720- CTGGCGGTCA ACAGCATGGC GGACTTGAGC AACTACAAAT GGGCGGGGTT CT - #ACAAGGAC 780- GCCAACTATG TTGCCTATGC CTCGCCGACC CAGAGTGCCG GCGACAAGGT GC - #TCAATATC 840- GGTTACGAAA ACGACCCGGT GTTCCGCGCG CTGGACGGCT CGTCGTTTAA CC - #TGTCGTCG 900- CTGGGCGTGC ACGACAAACC CCACGAGTCC ACCACCGATA ACATCGTCAG CT - #TCAACGAC 960- CACTACGCCT CGACGCTGTG GAATGTGCTG CCGTTTTCCA TCGTCAACCT GC - #CCACCTGG1020- GTCTCGCATT TGCCGACGGC GTACGGCGAT GGCATGACGC GCATCCTCGA GT - #CCGGCTTC1080- TACGACCAGA TGACCCGTGA CTCCACGGTG ATTGTTGCCA ACCTGTCCGA TC - #CGGCGCGG1140- GCCAACACCT GGGTGCAGGA CCTCAACCGC AATGCCGAGC CCCACAAGGG CA - #ACACGTTC1200- ATCATCGGCA GCGACGGCAA CGACCTGATC CAGGGCGGCA ACGGTGCGGA CT - #TTATCGAG1260- GGTGGCAAAG GCAACGACAC GATCCGCGAC AACAGCGGGC ACAACACCTT TT - #TGTTCAGC1320- GGCCACTTTG GCAATGATCG CGTGATTGGC TACCAGCCCA CCGACAAACT GG - #TGTTCAAG1380- GACGTGCAAG GAAGCACCGA CCTGCGTGAC CACGCGAAGG TGGTCGGCGC CG - #ATACGGTG1440- CTTACGTTTG GGGCCGACTC GGTGACGCTG GTCGGCGTGG GGCATGGCGG GC - #TGTGGACG1500- GAGGGCGTGG TGATCGGCTG ATTACTCACG CAACCGATCA GTGCCAGTGC TG - #CCCCCGCC1560#  1600            GGGC CGGTGGGGGT AGCCATAGCC- (2) INFORMATION FOR SEQ ID NO:4:-      (i) SEQUENCE CHARACTERISTICS:#pairs    (A) LENGTH: 1100 base     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (ii) MOLECULE TYPE: cDNA-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:- GGGCGATGGC TGGCAGGACG CGCCCCTCGG CCCCATCAAC CTGAGATGAG AA - #CAACATGA  60- AGAAGAAGTC TCTGCTCCCC CTCGGCCTGG CCATCGGCCT CGCCTCTCTC GC - #TGCCAGCC 120- CTCTGATCCA GGCCAGCACC TACACCCAGA CCAAATACCC CATCGTGCTG GC - #CCACGGCA 180- TGCTCGGCTT CGACAATATC CTCGGGGTCG ACTACTGGTT CGGCATTCCC AG - #CGCCTTGC 240- GCCGTGACGG TGCCCAGGTC TACGTCACCG AAGTCAGCCA GTTGGACACC TC - #GGAAGTCC 300- GCGGCGAGCA GTTGCTGCAA CAGGTGGAGG AAATCGTCGC CCTCAGCGGC CA - #GCCCAAGG 360- TCAACCTGAT CGGCCACAGC CACGGCGGGC CGACCATCCG CTACGTCGCC GC - #CGTACGTC 420- CCGACCTGAT GCCTTCCGCC ACCAGCGTCG GCGCCCCGCA CAAGGGTTCG GA - #CACCGCCG 480- ACTTCCTGCG CCAGATCCCA CCGGGTTCGG CCGGCGAGGC AGTCCTCTCC GG - #GCTGGTCA 540- ACAGCCTCGG CGCGCTGATC AGCTTCCTTT CCAGCGGCAG CGCCGGTACG CA - #GAATTCAC 600- TGGGCTCGCT GGAGTCGCTG AACAGCGAGG GGGCCGCGCG CTTCAACGCC AA - #GTACCCGC 660- AGGGCATCCC CACCTCGGCC TGCGGCGAAG GCGCCTACAA GGTCAACGGC GT - #GAGCTATT 720- ACTCCTGGAG CGGTTCCTCG CCGCTGACCA ACTTCCTCGA TCCGAGCGAC GC - #CTTCCTCG 780- GCGCCTCGTC GCTGACCTTC AAGAACGGCA CCGCCAACGA CGGCCTGGTC GG - #CACCTGCA 840- GTTCGCACCT GGGCATGGTG ATCCGCGACA ACTACCGGAT GAACCACCTG GA - #CGAGGTGA 900- ACCAGGTCTT CGGCCTCACC AGCCTGTTCG AGACCAGCCC GGTCAGCGTC TA - #CCGCCAGC 960- ACGCCAACCG CCTGAAGAAC GCCAGCCTGT AGGACCCCGG CCGGGGCCTC GG - #CCCCGGCC1020- CTTTCCCGGA AGCCCCCTCG CGTGAAGAAA ATCCTCCTGC TGATTCCACT GG - #CGTTCGCC1080#                 110 - #0- (2) INFORMATION FOR SEQ ID NO:5:-      (i) SEQUENCE CHARACTERISTICS:#pairs    (A) LENGTH: 1635 base     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (ii) MOLECULE TYPE: cDNA-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:- CAGGCCCCCA CGGCCGTTCT TAATGGCAAC GAGGTCATCT CTGGTGTCCT TG - #GGGGCAAG  60- GTTGATACCT TTAAGGGAAT TCCATTTGCT GACCCTCCTG TTGGTGACTT GC - #GGTTCAAG 120- CACCCCCAGC CTTTCACTGG ATCCTACCAG GGTCTTAAGG CCAACGACTT CA - #GCTCTGCT 180- TGTATGCAGC TTGATCCTGG CAATGCCATT TCTTGGCTTG ACAAAGTCGT GG - #GCTTGGGA 240- AAGATTCTTC CTGATAACCT TAGAGGCCCT CTTTATGACA TGGCCCAGGG TA - #GTGTCTCC 300- ATGAATGAGG ACTGTCTCTA CCTTAACGTT TTCCGCCCTG CTGGCACCAA GC - #CTGATGCT 360- AAGCTCCCCG TCATGGTTTG GATTTACGGT GGTGCCTTTG TGTTTGGTTC TT - #CTGCTTCT 420- TACCCTGGTA ACGGCTACGT CAAGGAGAGT GTGGAAATGG GCCAGCCTGT TG - #TGTTTGTT 480- TCCATCAACT ACCGTACCGG CCCCTATGGA TTCCTGGGTG GTGATGCCAT CA - #CCGCTGAG 540- GGTAACACCA ACGCTGGTCT GCACGACCAG CGCAAGGGTC TCGAGTGGGT TA - #GCGACAAC 600- ATTGCCAACT TTGGTGGTGA TCCCGACAAG GTCATGATTT TCGGTGAGTC CG - #CTGGTGCC 660- ATGAGTGTTG CTCACCAGCT TGTTGCCTAC GGTGGTGACA ACACCTACAA CG - #GAAAGAAG 720- CTTTTCCACT CTGCCATTCT TCAGTCTGGC GGTCCTCTTC CTTACTTTGA CT - #CTACTTCT 780- GTTGGTCCCG AGAGTGCCTA CAGCAGATTT GCTCAGTATG CCGGATGTGA TG - #CCAGCGCC 840- AGTGACAATG AAACTCTGGC TTGTCTCCGC AGCAAGTCCA GCGATGTCTT GC - #ACAGTGCC 900- CAGAACTCGT ACGATCTCAA GGACCTGTTT GGCCTGCTCC CTCAATTCCT TG - #GATTTGGT 960- CCCAGACCCG ACGGCAACAT TATTCCCGAT GCCGCTTATG AGCTCTACCG CA - #GCGGTAGA1020- TACGCCAAGG TTCCCTACAT TACTGGTAAC CAGGAGGATG AGGGTACTAT TC - #TTGCCCCC1080- GTTGCTATTA ATGCTACCAC GACTCCCCAT GTTAAGAAGT GGTTGAAGTA CA - #TTTGTAGC1140- GAGGCTTCTG ACGCTTCGCT TGATCGTGTT TTGTCGCTCT ACCCCGGCTC TT - #GGTCGGAG1200- GGTGCGCCAT TCCGCACTGG TATTCTTAAT GCTCTGACCC CTCAGTTCAA GC - #GCATTGCT1260- GCCATTTTCA CTGATTTGCT GTTCCAGTCT CCTCGTCGTG TTATGCTTAA CG - #CTACCAAG1320- GACGTCAACC GCTGGACTTA CCTTGCCACC CAGCTCCATA ACCTCGTTCC AT - #TTTTGGGT1380- ACTTTCCATG GTAGTGATCT TCTTTTCCAA TACTACGTGG ACCTTGGCCC AT - #CTTCTGCT1440- TACCGCCGCT ACTTTATCTC GTTTGCCAAC CACCACGACC CCAACGTTGG CA - #CCAACCTG1500- AAACAGTGGG ATATGTACAC TGATGCAGGC AAGGAGATGC TTCAGATTCA TA - #TGGTTGGT1560- AACTCTATGA GAACTGACGA CTTTAGAATC GAGGGAATCT CGAACTTTGA GT - #CTGACGTT1620#  1635- (2) INFORMATION FOR SEQ ID NO:6:-      (i) SEQUENCE CHARACTERISTICS:#pairs    (A) LENGTH: 1650 base     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (ii) MOLECULE TYPE: cDNA-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:- ATGGAGCTCG CTCTTGCGCT CCTGCTCATT GCCTCGGTGG CTGCTGCCCC CA - #CCGCCACG  60- CTCGCCAACG GCGACACCAT CACCGGTCTC AACGCCATCA TCAACGAGGC GT - #TCCTCGGC 120- ATTCCCTTTG CCGAGCCGCC GGTGGGCAAC CTCCGCTTCA AGGACCCCGT GC - #CGTACTCC 180- GGCTCGCTCG ATGGCCAGAA GTTCACGCTG TACGGCCCGC TGTGCATGCA GC - #AGAACCCC 240- GAGGGCACCT ACGAGGAGAA CCTCCCCAAG GCAGCGCTCG ACTTGGTGAT GC - #AGTCCAAG 300- GTGTTTGAGG CGGTGCTGCC GCTGAGCGAG GACTGTCTCA CCATCAACGT GG - #TGCGGCCG 360- CCGGGCACCA AGGCGGGTGC CAACCTCCCG GTGATGCTCT GGATCTTTGG CG - #GCGGGTTT 420- GAGGTGGGTG GCACCAGCAC CTTCCCTCCC GCCCAGATGA TCACCAAGAG CA - #TTGCCATG 480- GGCAAGCCCA TCATCCACGT GAGCGTCAAC TACCGCGTGT CGTCGTGGGG GT - #TCTTGGCT 540- GGCGACGAGA TCAAGGCCGA GGGCAGTGCC AACGCCGGTT TGAAGGACCA GC - #GCTTGGGC 600- ATGCAGTGGG TGGCGGACAA CATTGCGGCG TTTGGCGGCG ACCCGACCAA GG - #TGACCATC 660- TTTGGCGAGC TGGCGGGCAG CATGTCGGTC ATGTGCCACA TTCTCTGGAA CG - #ACGGCGAC 720- AACACGTACA AGGGCAAGCC GCTCTTCCGC GCGGGCATCA TGCAGCTGGG GG - #CCATGGTG 780- CCGCTGGACG CCGTGGACGG CATCTACGGC AACGAGATCT TTGACCTCTT GG - #CGTCGAAC 840- GCGGGCTGCG GCAGCGCCAG CGACAAGCTT GCGTGCTTGC GCGGTGTGCT GA - #GCGACACG 900- TTGGAGGACG CCACCAACAA CACCCCTGGG TTCTTGGCGT ACTCCTCGTT GC - #GGTTGCTG 960- TACCTCCCCC GGCCCGACGG CGTGAACATC ACCGACGACA TGTACGCCTT GG - #TGCGCGAG1020- GGCAAGTATG CCAACATCCC TGTGATCATC GGCGACCAGA ACGACGAGGG CA - #CCTTCTTT1080- GGCACCCTGC TGTTGAACGT GACCACGGAT GCCCAGGCCC GCGAGTACTT CA - #AGCAGCTG1140- TTTGTCCACG CCAGCGACGC GGAGATCGAC ACGTTGATGA CGGCGTACCC CG - #GCGACATC1200- ACCCAGGGCC TGCCGTTCGA CACGGGTATT CTCAACGCCC TCACCCCGCA GT - #TCAAGAGA1260- ATCCTGGCGG TGCTCGGCGA CCTTGGCTTT ACGCTTGCTC GTCGCTACTT CC - #TCAACCAC1320- TACACCGGCG GCACCAAGTA CTCATTCCTC CTGAAGCAGC TCCTGGGCTT GC - #CGGTGCTC1380- GGAACGTTCC ACTCCAACGA CATTGTCTTC CAGGACTACT TGTTGGGCAG CG - #GCTCGCTC1440- ATCTACAACA ACGCGTTCAT TGCGTTTGCC ACGGACTTGG ACCCCAACAC CG - #CGGGGTTG1500- TTGGTGAAGT GGCCCGAGTA CACCAGCAGC CTGCAGCTGG GCAACAACTT GA - #TGATGATC1560- AACGCCTTGG GCTTGTACAC CGGCAAGGAC AACTTCCGCA CCGCCGGCTA CG - #ACGCGTTG1620#         1650     TGTT CTTTGTGTAA__________________________________________________________________________