MXPA01011314A - Methods for producing plants with elevated oleic acid content - Google Patents

Abstract

By this invention, methods to produce oleic fatty acids in plant seed oils are provided. The methods of the present invention generally involve the suppression of a host plant cells endogenous&bgr;-ketoacyl-ACP synthase I protein. Also described in the instant invention are the plants, cells and oils obtained therefrom.

Description

METHODS TO PRODUCE PLANTS WITH ELEVATED OLEIC ACID CONTENT This application is a continuation in part of the application serial number 07 / 987,256 filed on December 7, 1992 which is a continuation in part of the application serial number 07 / 658,493 filed on August 15, 1990, now abandoned.

FIELD OF THE INVENTION The present invention is directed to a method for increasing the production of particular fatty acids in plants. In particular, the present invention is directed to a method for increasing oleic acid in plants.

INTRODUCTION BACKGROUND OF THE INVENTION Vegetable oils are used in a variety of industrial and edible uses. The novel vegetable oil compositions and / or the improved means for obtaining oily compositions, from biosynthetic sources or natural plants, are needed.

Depending on the use of the intended oil, several different fatty acid compositions are desired. For example, in some cases it would be useful to obtain a seed oil with a high ratio of oil to seed to obtain a desired oil at a low cost. This could be typical of a high-value oil product. In some cases, having a seed, oil with a lower ratio of oil to the seed could be useful to lower the caloric content. In other uses, edible vegetable oils with a higher percentage of unsaturated fatty acids are desired for reasons of cardiovascular health. And alternatively, temporary substitutes for highly saturated tropical oils such as palm oil and coconut oil may also find use in a variety of industrial and food applications. A postulated way to obtain said oils and / or modified fatty acid compositions is through the genetic engineering of the plants. However, in order to genetically engineer plants one must have the means to transfer genetic material to the plant in a stable and heritable manner. Additionally, one must have nucleic acid sequences capable of producing the desired phenotypic result, regulatory regions capable of directing the correct application of said sequences, and the like. Furthermore, it should be appreciated that in order to produce a desired phenotype it is required that the fatty acid synthase (FAS) pathway be modified to the extent that the ratios of reagents are modulated or changed. Higher plants seem to synthesize fatty acids via a common metabolic pathway. In developing seeds, where fatty acids are attached to skeletons of glycerol, formed triglycerides, are stored as a source of energy for subsequent germination, the FAS pathway is located in proplastidia. The first step involved is the formation of acetyl-ACP (acyl carrier protein) from acetyl-CoA and ACP catalyzed by the enzyme, acetyl-CoA: ACP transacylase (ATA). The elongation of acetyl-ACP to 16 and 18 carbon fatty acids involves the cyclic action of the following reaction sequences: condensation with a two-carbon unit from malonyl-ACP to form a β-ketoacyl-ACP (β-ketoacyl) -ACP synthase), reduction of keto function to an alcohol (β-ketoacyl-ACP reductase), dehydration to form an enoyl-ACP (β-hydroxyacyl-ACP dehydratase), and finally the reduction of enoyl-ACP to form acyl -ACC saturated elongate (enoyl-ACP reductase). β-ketoacyl-ACP synthase I, catalyzes the elongation of palmitoyl-ACP (C16: 0), where β-ketoacyl-ACP synthase II catalyses the final elongation towards steroyl-ACP (C18: 0). The common unsaturated fatty acids of plants, such as oleic, linoleic and a-linoleic acids are found in storage triglycerides, and originate from the desaturation of stearoyl-ACP to form oleyl-ACP (C18: 1) in a reaction catalyzed by a soluble plastid? -9 desaturase (also frequently referred to as "stearoyl-ACP desaturase"). Molecular oxygen is required for desaturation in which reduced ferredoxin serves as an electron co-donor. The additional desaturation is carried out sequentially by the actions of the desaturase? -12 and the desaturase? -15 attached to the membrane. These "desaturases" thus create mono or polyunsaturated fatty acids respectively. A third ß-ketoacyl-ACP synthase III has been reported in S. oleracea leaves that has specific activity towards very short acyl-ACP. This acetoacyl-ACP synthase or "β-ketoacyl-ACP" synthase III has a preference for acetyl-CoA over acetyl-ACP, Jaworski, J. G., et al., Plant Phys. (1989) 90: 41-44. It has been postulated that this enzyme may be an alternative route to start FAS, instead of ATA. By obtaining the nucleic acid sequences capable of producing a phenotype that results in FAS, the desaturation and / or incorporation of fatty acids into the glycerol backbone to produce an oil is subject to several obstacles including but not limited to the identification of metabolic factors of interest , choice and characterization of a source enzyme, with useful kinetic properties, purification of the protein of interest up to a level that will allow its amino acid sequencing, using the data of the amino acid sequence to obtain a nucleic acid sequence capable of using it as a probe to recover the desired DNA sequence, and the preparation of the constructions, transformation and analysis of the resulting plants.

Thus, the identification of target enzymes and plant sources useful for nucleic acid sequences of said target enzymes capable of modifying the nucleic acid compositions are needed. Ideally a white enzyme will be responsible for one or more applications alone or in combination with other nucleic acid sequences, in relation to increased / decreased oil production, the ratio of saturated to unsaturated fatty acids in the fatty acid pool, and / or to novel oily compositions as a result of modifications of the fatty acid group. Once the target enzyme (s) is identified and classified, the amounts of purified protein and purification protocols are needed for sequencing. Finally, useful nucleic acid constructs having the necessary elements to provide a phenotypic modification and the plants containing said constructions are needed.

BRIEF DESCRIPTION OF THE INVENTION The present invention is directed to methods for producing to plant methods high levels of oleic acid (C18: 1) as a percentage of total fatty acids. The method generally comprises growing a plant having a construct having a component operably linked in the 5 'to 3' direction of the transcript, a functional promoter region in a host plant cell, and at least a portion of a nucleic acid sequence that encodes a β-ketoacyl ACP in an antisense orientation and a transcription determination sequence. The methods described herein are used to produce plants with increased levels of oleic acid. The increments of at least 5% to 60%, preferably, 10% to 50%, more preferably 10% to 40% on the wild-type seed oil are covered by the methods provided in the present invention. In one embodiment of the present invention, Brassica seed oil having increased oleic acid is obtained using the methods of the present invention. The oleate content from the Brassica seed oil preferably comprises more than 65%, more preferably more than about 75% of the fatty acid portions in the oil. The oil of the present invention can be used as a source mixture to make a mixed oily product, or it can be used in food preparation. In another embodiment of the present invention, a Brassica oil having a decreased polyunsaturated fatty acid composition is obtained using the methods described herein. Brassica oils with polyunsaturated fatty acid compositions of less than about 12 weight percent are exemplified herein.

DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, the constructs and methods are provided for the production of plants with an increased level of oleic acid (C18: 1), as a percentage of the total fatty acids. Methods for producing said plants generally comprise transforming a host plant cell with expression constructs having a functional promoter sequence in a plant operatively associated with at least a portion of a nucleic acid sequence encoding a β-ketoacyl-ACP synthase (referred to hereinafter referred to as KAS) in an antisense orientation, and a transcription termination sequence. The expression constructs provide a novel method for increasing the levels of oleic acid in the seed oil of the transformed plants. Β-ketoacyl-ACP synthases are well known in the art for their performance in fatty acid biosynthesis. The first step in the fatty acid biosynthesis is the formation of acetyl-ACP (acyl carrier protein) from acetyl-CoA and ACP catalyzed by a short-chain preferential condensing enzyme, β-ketoacyl-ACP synthase (KAS) III . The elongation of acetyl-ACP to fatty acids of 16 and 18 carbons implies the cyclic action of the following sequence of reactions: condensation with a two-carbon unit from malonyl-ACP to form a longer β-ketoacyl-ACP ( β-ketoacyl-ACP synthase), reduction of keto function to an alcohol (β-ketoacyl-ACP reductase), dehydration to form an enoyl-ACP (β-hydroxyacyl-ACP dehydratase), and finally reduction of enoyl-ACP to form saturated elongated acyl-ACP ( enoyl-ACP reductase), β-ketoacyl-ACP synthase I (KAS I), is mainly responsible for the elongation of palmitoyl-ACP (C16: 0), while β-ketoacyl-ACP synthase II (KAS II) is mainly responsible of the final elongation to stearoyl-ACP (C18: 0). The genes encoding the peptide components from β-ketoacyl-ACP synthase I and II have been cloned from a number of higher plant species, including castor (Ricinus communis) and Brassica species (USPN 5,510,255). KAS I activity was associated with a si protein synthase factor that has an approximate molecular weight of 50 kD (factor B synthase) and KAS II activity was associated with a combination of two protein synthase factors, the B-factor synthase. 50 kD and a 46 kD protein designated factor A synthase. The cloning and sequence of a plant gene encoding a KAS II protein has been reported by Tai and Jaworski. { Plant Physiol, (1993) 703: 1361-1367). Surprisi, it has been found herein that the antisense expression of at least a portion of a KAS sequence in the seed cells of a host plant cell increases the oleic acid content of the seed oil. Preferably, the KAS sequences used in the present invention are derived from the endogenous KAS sequence of the white host plant, also referred to herein as the native KAS sequence. The person skilled in the art will recognize that also non-native KAS sequences obtained from sources different from that of the white host plant are also useful in the present invention. By white host plant it refers to the plant within which the expression constructs containing the KAS sequences are transformed. As described in more detail in the examples below, a β-ketoacyl ACP synthase type I (referred to herein as KASI) coding sequence from Brassica (U.S. Patent No. 5,475,099, and 5,510,255, all of which incorporated herein by reference) is used in the construction of expression in an antisense orientation to generate transgenic Brassica plants with decreased KASI production in the host cells. Surprisi, it has been demonstrated herein that the transformation of a plant with a construct that provides antisense expression of the KASI gene leads to a significant increase in the levels of oleic acid (C18: 1) obtained as a percentage of the total fatty acids produced in the seed oil In addition, the transformed seeds demonstrated altered compositions of polyunsaturated fatty acids as a result of the expression of KASI antisense, as seen in the decrease in linoleic acid (C18: 2) and linoleic acid (C18: 3) observed in the oilseed seeds of plants which contain high oleic acid. Therefore, by using the methods of the invention, seeds are provided which produce an altered composition of fatty acids and a vegetable oil ratio which has an increased content of oleic acid and a decreased content of linoleic and linolenic acid. Therefore, the transformed seeds can provide a source of modified seed oil. Constructs used in the methods of the present invention may also find use in plant genetic engineering applications in conjunction with plants containing high levels of fatty acids oleates (18: 1) to further increase the levels of oleic acid. Said plants can be obtained by the expression of staroyl-ACP desaturases sequences, such as those sequences described by Knutzon et al. (Proc. Nat. Acad. Sci. (1992) 89: 2624-2628). further, plants containing increased levels of oleic acid can be obtained by nucleic acid sequences that are expressed by suppressing the endogenous? 12 desaturases. Such sequences are known in the art and are described in PCT publication WO 94/11516. The increase in oleic acid can also be obtained by the suppression of? 12-desaturases and? -15 desaturases, such as the methods taught in the patent of E.U.A. No. 5,850,026. Plants that produce a high content of oleic acid can also be obtained by conventional mutation and plant cross-breeding programs. Said methods for mutation are known in the art and are described, for example, in the patent of E.U.A. No. 5,625,130.

In addition, constructs and methods for increasing oleic acid in seed oil can also find their use in plant genetic engineering applications in conjunction with plants containing decreased levels of fatty acids linoleates (C18: 2) and / or linolenate ( 18: 3). Said plants with high stearate levels and / or with decreased levels of linoleate and / or linolenate can also be obtained through genetic engineering, or by conventional mutation and plant cross-breeding programs. For example, methods for increasing the stearate content from a seed oil are known in the art and are described for the use of a thioestearase from mangosteen (Garcinia mangostana), Garm FatA1 (Hawkins and Kridl (1998)). Plant Journal 13 (6): 743-752, and PCT patent application WO 96/36719. In addition, constructs and methods for increasing oleic acid in seed oil can also find their use in plant genetic engineering applications in conjunction with plants containing increased amounts of medium chain fatty acids to further increase the medium chain fatty acid content of the resulting plants.These plants with high levels of chain fatty acids can be obtained through genetic engineering, or by conventional mutation and cross-breeding programs, methods to increase medium-chain fatty acids by genetic engineering are in the art, and are described, for example, in the US patents. Nos. 5,455,167, and 5,512,482 and in WO98 / 46776. Therefore, designed recombinant constructs that have the sequence KASI in a reverse orientation for the expression of an antisense sequence or the use of co-suppression, also known as "trans-switch", the constructions are found their use in the methods of the present invention. Antisense methods are well known in the art, and are described, for example, by Van der Krol, et al. (1988) Biotechniques 6: 958-976; Shechy, I went to. (1988) Proc. Nati Acad. Sci. USA 85: 8805-8809; Cannon, I went to. (1990) Plant Molec Biol. 15: 39-47. Methods. Methods for sense suppression are well known in the art, and are described, for example, by Napoli et al. (1990) Plant Cell 2: 279-289; van der Krol, e to al. (1990) Plant Cell 2: 291-299; and Smith, I went to. (1990) Mol. Gen Genetics 224: 477-481. Other methods for the suppression of native expression of target sequences are also known in the art, and include, but are not limited to, nucleic acid molecules with RNA breaking activity, referred to as ribozymes (described in PCT publication WO 97). / 10328), as well as antisense and sense suppression statements, such as those taught by Waterhouse, et al. (1998) Proc. Nati Acad. Sci. USA 95: 13959-13964. Therefore, by suppressing the endogenous fatty acid biosynthesis system, for example by means of the methods of the present invention, a reduction in the amounts of phosphatidyl choline (also referred to as PC) can be obtained. Such reductions in PC result in a lower substrate level for the subsequent desaturase leading to increased amounts of monounsaturated fatty acids. The sequences that are in an antisense orientation can be found in cassettes that at least provide the transcription of the sequence encoding the synthase. By anti-sense it is meant to mean a DNA sequence in the 5 'to 3' direction of the transcription which encodes a sequence complementary to the sequence of interest. It is preferred that an "antisense synthase" is complementary to a plant synthase gene endogenous to the host plant. Any promoter capable of carrying out expression in a host plant that causes the initiation of high levels of transcription in all senses of storage during the development of the seed is sufficient. Seed-specific promoters may be desirable. To prepare the expression constructions, the various DNA fragments can be manipulated, so that they are provided for the DNA sequence in the proper orientation and, as appropriate in the appropriate reading frame. Towards this end, adapters or couplers can be used to join the DNA fragments or other manipulations can be involved to provide convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, coupling, resection, ligation or the like can be employed, where the insertions, of lesions or substitutions, for example transitions and transversions, may be involved. For the most part, the constructions will involve functional regulatory regions in plants that provide modified production of vegetable KAS I, and modification of the fatty acid composition. The open reading frame, which codes for plant KAS I or the functional fragment thereof will be attached to its 5 'end in a regulatory region of the start transcript such as in the wild-type sequence which is normally found 5' of the structural gene thioesterase, or in the heterologous regulatory region from a gene that is naturally expressed in plant tissues. Examples of useful plant regulatory gene regions include those from T-DNA genes, such as nopalin or octopin synthase plant virus genes, such as CaMV 35S, or from native plant genes. For such applications when the non-coding regions towards 5 'are obtained from other genes that are regulated during the maturation of the seed, preferentially those that are expressed in the plant embryonic tissue, such as ACP, napin and the 7S subunit of ß -conglicillinin (Chen et al., (1986), Proc. Nati. Acad. Sci. 83: 8560-8564), control regions of the start of transcription, as well as the Lesquerella hydroxylase promoter (described in Broun, et al. (1998) Plant Journal 13 (2): 201-210 and in the US patent application Serial No. 08 / 898,038) and the stearoyl-ACP desaturase promoter (Slocombe, et al. (1994) Plant Physiol. 104: 1167-1176), as desired. Said "seed-specific promoters" may be obtained and used in accordance with the teachings of USPN 5,420,034 which have a title "Seed-Specific Transcriptional Regulation" and in Chen et al. (1986), Proc. Nati Acad. Sci. 83: 8560-8564. The regions of initiation of transcription that are preferably expressed in plant tissue, ie, which are not detected in other plant parts, are considered desirable for fatty acid modifications in order to minimize any disruptive or adverse effect of the gene product. . The regulatory transcription termination regions can be provided in DNA constructs of this invention as well. The transcription termination regions can be provided by the DNA sequence encoding KAS I or a convenient transcription determination region derived from a different gene source, eg, the transcription determination region that is naturally associated with the transcription region. region of the beginning of transcription. The person skilled in the art will recognize that any convenient transcription termination region that is capable of terminating transcription in a plant cell can be employed in the constructions of the present invention. As described herein, transcription determination sequences derived from DNA sequences preferably expressed in plant seed cells are employed in the expression constructs of the present invention.

The transformation method is not critical to the present invention; Several methods of plant transformation are currently available. With new methods available to transform crops, these can be applied directly in the present. For example, many plant species naturally susceptible to infection by Agrobacterium can successfully transform the tripartite or binary vector methods of Agrobacterium-mediated transformation. In addition, the techniques of microinjection, bombardment of DNA particles, and electroporation have been developed which allow the transformation of several species of monocotyledonous and dicotyledonous plants. To develop the DNA construct, the various components of the construct or fragments thereof will normally be inserted into a convenient cloning vector which is capable of replication in a bacterial host, e.g., E. coli. Numerous vectors exist that have been described in the literature. After each cloning, the plasmid can be isolated and subjected to further manipulation, such as restriction, insertion of new fragments, ligation, deletion, insertion, resection, etc., so that the components of the desired sequence are coupled. Once the construction has been completed, it can then be transferred to an appropriate vector for further manipulation in accordance with the transformation of the host cell.

Normally, included with the DNA construct will be a structural gene that has the regulatory regions necessary for expression in a host and to provide for the selection of transforming cells. The gene can be provided for resistance to a cytotoxic agent, for example, antibiotic, heavy metal, toxin, etc., by providing phototrophy complementation to an auxotrophic host, viral immunity or the like. Depending on the number of different host species in which the expression construct or the components thereof are introduced, one or more markers may be employed, wherein the different conditions for selection are used for the different hosts. A number of labels have been developed to be used for the selection of transformed plant cells, such as those that provide resistance to various antibiotics, herbicides, or the like. The particular marker employed is not essential for this invention, one or the other marker is preferred depending on the particular host and the manner of construction. As mentioned above, I believe that the construction of DNA is introduced into the plant host is not critical to this invention. Any method that provides efficient transformation can be used. Various methods of transforming plant cells include the use of Ti or Ri plasmids, microinjection, electroporation, particle bombardment of DNA, fusion by liposome, or the like. In many cases, it will be desirable to have the construction bounded on one or both sides by T-DNA, particularly having the left and right boundaries, more particularly the right boundary. This is particularly useful when the construction uses A. tumefaciens or A. rhizogenes as a mode for transformation, although the T-DNA limits may find their use with other modes of transformation. Several methods to transform soy cells have been previously described. Examples of soy transformation methods have been described, for example, by Christou et al. patent of E.U.A. No. 5,015,380 and by Hinchee eí al. patent of E.U.A. No. 5,41,011, all of which is incorporated herein by reference. Once a transgenic plant is obtained which is capable of producing seeds having a modified fatty acid composition, traditional plant cross-linking techniques, including mutagenesis methods, can be employed for further manipulation of the fatty acid composition. Alternatively, the additional external DNA sequence that modifies the fatty acid can be introduced via genetic engineering to further manipulate the fatty acid composition. One may choose to provide the transcription or transcription and translation to one or more different sequences of interest in conjunction with the expression of a stearoyl-ACP thioesterase from plants in a plant host cell. In particular, the reduced expression of stearoyl-ACP desaturase in combination with the expression of a plant stearoyl-ACP thioesterase may be preferred in some applications.

When one wishes to provide a transformed plant for the combined effect of more than one nucleic acid sequence of interest, typically a separate nucleic acid expression construct will be provided for each. The constructions, as described above, contain regions of regulatory control of transcription or of transcription and translation. The constructs can be introduced into the host cells by the same or different methods, including the introduction of said trait by including two transcription cassettes in a single transformation vector the simultaneous transformation of two expression constructs, retrotransformation using plant tissue that expresses a construction with an expression construction for the second gene, or by crossing transgenic plants via traditional plant crossing methods, as long as the resulting product is a plant that has both characteristics integrated into its genome. By decreasing the amount of stearoyl-ACP desaturase, an increased percentage of saturated fatty acid is provided. Using antisense, trans-switch, ribozyme or other reducing technology of stearoyl-ACP desaturase, a decrease in the amount of ß-ketoacyl-ACP synthase available by the plant cell occurs, resulting in a higher percentage of oleate fatty acids. Of special interest is the production of triglycerides that have increased levels of oleic acid. In addition, the production of a wide variety of oleate is desired. Therefore, plant cells having decreased or higher levels of oleate fatty acids produced by the methods described herein are contemplated. For example, fatty acid compositions, including oils, having a 65% oleate level as well as compositions designed to have more than about 78% oleate level or other fatty acid composition (s) are also contemplated. modified. The seeds of the invention that have been transformed with the constructions that provide KASI expression provide a source for new oily compositions. The use of such constructions, for example, results in the substantial increase in the content of oleic acid in the seed oil. By the substantial increase an oleic acid increase of at least about 65% of the total fatty acid species is intended. Therefore, the seeds of the invention that have been transformed with an antisense KASI expression construct provides a source for modified oils having a high content of oleic acid. The content of oleic acid in any seed can be altered by the present methods, even those seeds which have a high content of oleic acid naturally. The alteration of the seeds having high oleic acid contents naturally by the present methods can result in total oleic acid content greater than about 78%.

Importantly, there is also a decrease in the linoleic acid and linolenic content. By decreasing the linoleic fatty acid content, a decrease in the linoleic acid species of less than about 15 mole percent of the total fatty acid species is intended. By decreasing the linolenic fatty acid content, a decrease in linolenic acid of approximately less than 7 mole percent of the total fatty acid content of the seed oil is intended. Therefore, the methods of the invention result in oils that are more oxidatively stable than naturally occurring oils. The modified oils of the invention are oils with high oleic content and low linoleic content, of low saturation. In addition, the present invention provides high oils in monounsaturated fatty acids which are important as a constituent of the diet. Based on the methods described in this, the seed oil can be modified to design an oil with a high content of oleic acid. Oils with high oleic acid content could have a longer shelf life as well as the oleic acid content could lead to stability. The methods of the present invention comprise the use of expression or plant transcription constructs having a plant ß-ketoacyl-ACP synthase as the DNA sequence of interest can be employed with a wide variety of plant life, particularly, plant life involved in the production of vegetable oils for edible and industrial uses. More especially preferred are harvests of oiled temperate seeds. Plants of interest include, but are not limited to, rapeseed (Cañóla and high ic acid varieties), sunflower, safflower, cotton, Cuphea, soybean, peanut, flax, coconut, palm oil and corn. Depending on the method for introducing the recombinant constructs into the host cell, other DNA sequences may be required. Importantly, this invention applies to similar dicotyledonous and monocotyledonous species and will be readily applicable to novel and / or improved transformations and regulatory techniques. The oil having an increased content of oleic acid can be processed using methods well known in the art. In addition, the processed oil having increased oleic acid produced by the methods of the present invention finds use in a wide variety of end uses, such as edible as well as industrial uses. Said applications include, for example, salad oils, frying oils, cooking oils, sprinkled oils, and viscous applications for food products. The oil obtained according to the methods of the present invention has a greater oxidative stability, due to the increase of monounsaturated fatty acid and to the reduction in the content of polyunsaturated fatty acid, thus reducing the need for chemical modifications, such as hydrogenation. The invention which has now been generally described will be more readily understood as reference to the following examples which are included for purposes of illustration only and are not intended to limit the present invention.

EXAMPLES EXAMPLE 1 Expression constructions A construct containing the cDNA sequence of Brassica campestris factor B synthase (also referred to as KAS I) (SEQ ID NO: 1), pCGN3248 (described in U.S. Patent No. 5,475,099, the entirety of which is incorporated herein by reference), is mutagenized to insert the Smal, BglII, and SalI restriction sites at approximately 200 bases up to 3 'of the Stop signal of the translation, resulting in pCGN3255. pCGN3255 is digested and the internal B factor cDNA from the SalI site located at approximately 140 bases from the 5 'end of the cDNA and the 3' site of BglII inserted by mutagenesis. The resulting B-factor cDNA fragment from the resulting synthase is ligated into pCGN3223, digested with BglII and SalI, the napin expression cassette described above, results in the pCGN3257 antisense construct. Therefore, the transcription of the factor sequence B of Brassica synthase from the napin promoter will result in the production of one strand of mRNA that is complementary to that of the endogenous Brassica synthase factor B gene.

The fragment containing the gene synthase in the expression cassette, sequences / synthase / 3 'sequences, can be cloned into a binary vector as described by McBride and Summerfeit (Pl. Mol. Biol. (1990) 14: 269-276 ) for processing by Agrobacterium. Other binary vectors are known in the art and can also be used for synthase cassettes. For example, the construction of the Brassica antisense synthase factor B in a napin expression cassette, pCGN3257 is digested with Asp718 (same recognition sequence as Kpnl) and cloned into pCGN1578 digested with As? d (McBride and Summerfeit, previously mentioned) giving the binary construction pCGN3259. Transformed Brassica napus plants containing the constructions described above are obtained as described in Radke et al., (Theor. Appl. Genet. (1988) 75: 685-694 and Plant Cell Reports (1992) 11: 499-505 ).

EXAMPLE 2 Analysis of fatty acids The fatty acid composition is analyzed from 50 individual seeds from the two lines. The fatty acid compositions are shown in Table 1, which refers to the fatty acid composition from about 50 individual seeds from two plant lines containing the pCGN3259 construct.

TABLE 1 (CONTINUED) 34 3259-D-5 0.01 0.08 4.47 0.12 1.46 62.06 20.79 8.85 0.5 1.36 0.08 0.21 0 or 35 3259-D-5 0.01 0.09 4.33 0.12 1.07 49.48 29.88 13.11 0.34 1.31 0.11 0.1 0.01 or 36 3259- D-5 0.01 0.07 3.99 0.08 1.85 64.37 19.67 7.5 0.6 1.41 0.08 0.35 0 or 37 3259-D-5 0.01 0.09 4.21 0.16 2.62 60.45 22.54 7.54 0.76 1.27 0.06 0.3 0 or 38 3259-D-5 0.01 0.1 4.99 0.28 1.19 50.64 27.81 13.04 0.43 1.19 0.1 0.21 0 or 39 3259-D-5 0.01 0.09 3.7 0.06 2.51 70.93 15.04 5.1 0.73 1.36 0.1 0.36 0 or 40 3259-D-5 0.02 0.07 4.2 0.12 2.21 63.71 20.51 7.06 0.64 1.16 0.06 0.26 0 or 41 3259- D-5 0.03 0.07 4.56 0.1 4.58 67.39 16.77 4.1 1.16 1.22 0 0 0 or 42 3259-D-5 0.02 0.11 5.68 0.36 0.98 38 34.92 16.02 0.38 1.09 0.12 0.23 0.01 0.01 43 3259-D-5 0.02 0.08 4.33 0.14 1.47 57.52 26.51 8.03 0.44 1.27 0.09 0.11 0 or 44 3259-D-5 0.01 0.1 4.61 0.16 1.21 42.24 37.08 13.22 0.36 0.76 0.15 0.09 0 or 45 3259-D-5 0.01 0.1 4.43 0.17 1.91 65.33 21.13 4.51 0.66 1.44 0.07 0.24 0 or 46 3259-D-5 0.01 0.08 4.28 0.14 1.2 62.88 18.83 10.37 0.5 1.42 0. 0.27 0.01 or 47 3259-D-5 0.02 0.08 3.71 0.08 1.4 63.62 21.07 7.97 0.46 1.33 0.12 0.14 0.01 or 48 3259-D-5 0.01 0.07 4.45 0.12 2.02 58.81 24.02 8.35 0.69 1.11 0.08 0.26 0 or 49 3259-D-5 0.01 0.08 4.04 0.1 1.88 60.85 22.09 8.83 0.57 1.08 0.1 0.27 0 or 50 3259-D-5 0.02 0.12 4.79 0.17 1.55 56.32 29.83 5.19 0.54 1.12 0.12 0.24 0 or 51 3259-D-12 0.01 0.06 4.02 0.09 1.57 70.55 12.5 8.61 0.69 1.4 0.06 0.43 0 or • -a 52 3259-D-12 0.01 0.07 4.36 0.09 1.96 66.33 17.81 6.73 0.84 1.21 0.06 0.52 or or 53 3259-D-12 0.01 0.06 4.19 0.09 2.08 67.2 15.17 8.27 0.88 1.46 0.07 0.51 oo 54 3259-D-12 0.01 0.12 4.73 0.09 1.46 68.84 12.97 8.87 0.7 1.69 0.08 0.44 oo 55 3259-D-12 0.01 0.09 4.17 0.08 1.65 65.93 17.54 7.78 0.75 1.45 0.07 0.44 0.01 or 56 3259-D-12 0.01 0.07 3.96 0.09 1.33 68.07 14.26 9.93 0.57 1.35 0.07 0.3 oo 57 3259-D-12 0.01 0.06 3.88 0.09 1.55 73.99 10.14 7.65 0.69 1.5 0.05 0.39 oo 58 3259-D-12 0.01 0.06 3.7 0.07 1.7 73.25 10.49 8.04 0.74 1.48 0.05 0.42 0.01 or 59-3259-D-12 0.01 0.07 4.01 0.07 2.55 70.82 13.96 5.48 1.02 1.49 0 0.52 oo 60 3259-D-12 0.01 0.06 3.81 0.07 1.72 73.95 10.7 6.85 0.71 1.67 0.05 0.38 0.01 or 61 3259-D-12 0.01 0.07 3.66 0.06 1.63 73.87 9.29 8.71 0.72 1.52 0.05 0.4 oo 62 3259-D-12 0.01 0.06 3.72 0.07 2 74, 08 11.24 5.89 0.8 • 1.63 0.07 0.43 oo 63 3259-D-12 0.01 0.07 4.16 0.08 1.97 70.59 13.03 7.03 0.89 1.61 0.06 0.53 oo 64 3259-D-12 0.01 0.06 3.7 0.05 2.36 76.06 8.56 6.05 0.97 1.62 0.05 0.49 oo 65 3259 -D-12 0.01 0.06 3.78 0.07 2.53 75.32 .9.63 5.15 1.04 1.78 0.05 0.56 oo CUADR01 (CONTINUED) 66 3259-D-12 0.02 0.07 3.97 0.05 2.98 74.17 10.54 4.54 1.2 1.84 0.03 0.6 0 0 67 3259-D-12 0.01 0.08 4.32 0.13 1.47 65.8 15.69 9.73 0.71 1.49 0.09 0.48 0 0 68 3259-D-12 0.01 0.08 3.93 0.08 1.63 63.93 18.08 9.31 0.73 1.73 0.09 0.38 0 0 69 3259-D-12 0.01 0.1 4.32 0.16 1.55 57.94 18.89 14.52 0.68 1.37 0.09 0.37 0 0 70 3259-D-12 0.01 0.07 3.74 0.05 2.93 76.68 8.48 4.56 1.26 1.56 0.03 0.62 0.01 0 71 3259-D-12 0.01 0.07 3.71 0.06 2.38 74.15 11.3 5.12 0.94 1.73 0 0.5 0.01 0.01 72 3259-D-12 0.02 0.07 4.27 0.09 2.04 66.68 15.27 8.81 0.81 1.41 0.06 0.45 0.01 0 73 3259-D-12 0.01 0.06 3.86 0.06 2.65 73.79 10.34 5.98 1.06 1.59 0.06 0.53 0 0 74 3259-D-12 0.02 0.09 4.42 0.19 1.83 62.32 19.81 8.64 0.72 • 1.53 0.06 0.37 0 0 75 3259-D-12 0.01 0.06 • 3.92 0.08 2.28 71.48 12.62 6.77 0.9 1.38 0.05 0.44 0 0 76 3259-D-12 0.02 0.1 4.28 0.09 1.62 66.55 16.75 8.04 0.72 1.39 0.05 0.38 0.01 0 77 3259-D-12 0.02 0.12 5.26 0.29 1.08 48.95 25.88 16.1 0.45 1.45 0.1 0.26 0 0 78 3259-D-12 0.01 0.07 4.04 0.07 1.8 70.21 12.59 8.71 0.78 1.37 0.04 0.3 0 0 79 3259-D-12 0.01 0.08 4.12 0.09 2.15 66.19 16.51 7.74 0.89 1.67 0.06 0.5 0 0 80 3259-D-12 0.01 0.07 4.01 0.06 2.41 73.08 11.19 5.77 0.99 1.79 0.06 0.54 0.01 81 3259-D-12 0.02 0.08 4.1 0.1 2.53 69.78 13.3 7.04 1.02 1.41 0.08 0.54 0 0 82 3259-D-12 0.01 0.07 3.96 0.07 1.86 67.91 14.86 8.65 0.77 1.36 0.08 0.41 0 0 83 3259-D-12 0.01 0.08 3.86 0.11 2.62 70.46 14.3 5.71 0.96 • 1.38 0.05 0.44 0.02 0 w 00 84 3259-D-12 0.01 0.07 3.97 0.07 2.21 72.94 11.03 6.93 0.94 1.32 0 0.51 0 0 85 3259-D-12 0.01 0.09 4.56 0.06 2.21 75.49 9.07. 5.13 1.03 1.58 0.06 0.69 0 0 86 3259-D-12 0.01 0.08 4.31 0.07 2.38 72.79 11.07 6.1 1.04 1.47 0.08 0.58 0 0.01 87 3259-D-12 0.01 0.07 4.27 0.09 2.04 68.97 13.13 8.5 0.9 1.44 0.06 0.52 0 0 88 3259-D-12 0 89 3259-D-12 0.01 0.06 3.8 0.07 1.91 75.27 8.94 7.02 0.82 1.61 0 0.48 0 0.01 90 3259-D-12 0.01 0.07 4.11 0.08 1.76 71.23 13.1 6.92 0.77 1.48 0.06 0.41 0 0 91 3259-D-12 0.01 0.07 3.82 0.06 1.93 74.41 10.25 6.32 0.86 1.76 0 0.5 0 0 92 3259-D-12 0.01 0.08 4.37 0.07 2.7 72.33 11.04 6.12 1.11 1.52 0.05 0.58 0 0 93 3259-D-12 0.01 0.1 '4.35 0.12 2.04 62.74 20.05 7.78 0.84 1.36 0.07 0.53 0 0 94 3259-D-12 0.01 0.08 4.25 0.15 1.87 63.69 14.92 12.64 0.71 1.28 0.07 0.31 0 0 95 '3259-D-12 0.01 0.07 3.88 0.06 3.67 69.57 12.16 6.9 1.3 1.67 0.06 0.63 0.02 0 96 3259-D-12 0.01 0.08 4.15 0.1 1.52 70.42 13.73 7.38 0.68 1.5 0.06 0.37 0.01 0 97 3259-D-12 0.01 0.1 3.92 0.05 1.74 77.86 8.39 5.02 0.74 1.79 0.03 0.35 0 0 98 3259-D-12 0.01 0.07 3.98 0.07 3.31 68.15 13.87 7.33 1.15 1.38 0.06 0.6 0 0 TABLE 1 (CONTINUED) 99 3259-D-12 0.01 0.07 4.08 0.1 1.52 71.53 12.83 7.07 0.68 1.61 0.05 0.44 0 100 3259-D-12 0.02 0.08 4.03 0.05 2.79 78.31 7.79 3.47 1.13 1.72 0.05 0.57 0 t \ so) The results of the fatty acid composition analysis demonstrate a significant increase in oleic acid (18: 1) which is obtained in the Brassica seed oil containing the KAS I antisense expression construct. Oleic acid levels as high as at least 78 mol percent are obtained using such constructions, for example in lines 3259-D12 (# 100). Minor increases are also obtained, for example several lines are obtained that contain approximately 70 mole percent of oleic acid. In addition, most of the lines obtained contain more than about 65 mole percent of oleic acid. In addition, reductions in the amount of polyunsaturated fatty acids are obtained in the seed oil from the Brassica plants containing KASI antisense expression constructs. No amounts of linoleic acid are decreased below 15% of the total fatty acid species, and as low as about 7.8 mol% in at least one seed of 3259-D-12. The content of linolenic acid in the seeds of Brassica plants containing KASI antisense expression constructs is reduced to less than about 6 mole percent, and in some lines the content of linolenic acid is reduced to about 5.1 mole percent. Total polyunsaturated fatty acid levels less than about 13 mole percent can be obtained using such constructions.

EXAMPLE 3 Identification of soybean ß-ketoacyl-ACP synthase I sequences In order to produce soybean lines with increased oleic acid content, additional sequences of KASI DNA from the soy EST library were identified. Four EST sequences from soy were identified which were related to the Brassica KASI sequence (U.S. Patent No. 5,475,099) (SEQ ID NOs: 2-5). Nine EST sequences were also identified in the EST soy libraries which are related to the Brassica KASII sequence (SEQ ID NO: 6-14). To obtain the complete coding region corresponding to the soy KAS I EST sequences, the synthetic oligonucleotide primers were designed to amplify the 5 'and 3' ends of the partial cDNA clones containing the sequences related to acyltransferase. The primers are designed in accordance with the respective KAS I EST sequence of soybeans and are used in the rapid amplification reactions of cDNA ends (RACE) (Frohman et al. (1998) Proc. Nati. Acad. Sci. USA 85: 8998-9002) using the Marathon cDNA amplification kit (Clontech Laboratories Inc., Palo Alto, CA). Once the DNA sequence corresponding to the entire coding sequence, several portions of the sequence, or the entire coding sequence can be used for the construction of antisense expression vectors for use in transformed soybean plants using methods as provided in the present invention. All publications and patent applications mentioned in this specification are indicative of the skill level of those skilled in the art to which this invention pertains. All publications and patent applications are hereby incorporated by reference to the extent as if they were individual publications or patent applications specifically and individually indicated for incorporation as references. Although the foregoing invention has been described in detail by means of illustrations and examples for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (30)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for increasing the content of oleic acid from a host plant cell comprising: growing a plant having an introduced nucleic acid construct, said construct comprised in the 5 'to 3' direction of the transcription, a promoter functional in a host plant cell, a nucleic acid sequence for a β-ketoacyl-ACP-synthase I protein, and a functional transcription determination region in a plant cell, wherein the transcription of said nucleic acid sequence suppresses the expression of endogenous ß-ketoacyl-ACP-synthase I.

2. The method according to claim 1, further characterized in that said nucleic acid sequence is in antisense orientation with respect to said promoter.

3. The method according to claim 1, further characterized in that said nucleic acid sequence is in sense orientation with respect to said promoter.

4. The method according to claim 1, further characterized in that said nucleic acid sequence is ribozyme.

5. - The method according to claim 1, further characterized in that said sequence of β-ketoacyl-ACP-synthase I is obtained from the endogenous host plant.

6. The method according to claim 4, further characterized in that said endogenous host plant is selected from the group consisting of Brassica, soybeans and corn.

7. The method according to claim 1, further characterized in that said host plant cell is a seed cell.

8. A seed oil obtained according to the method according to claim 1, further characterized in that said content of oleic acid comprises more than about 65 mole percent.

9. A method for decreasing the polyunsaturated fatty acid content of a host plant cell comprising: growing a plant having an introduced nucleic acid construct, said construction comprised in the 5 'to 3' direction of the transcript, a functional promoter in a host plant cell, a nucleic acid sequence for a β-ketoacyl-ACP-synthase I protein, and a functional transcription determination region in a plant cell, wherein the transcription of said nucleic acid sequence suppresses the expression of endogenous ß-ketoacyl-ACP-synthase I.

10. - The method according to claim 9, further characterized in that said nucleic acid sequence is in antisense orientation with respect to said promoter.

11. The method according to claim 9 further characterized in that said nucleic acid sequence is in sense orientation with respect to said promoter.

12. The method according to claim 9, further characterized in that said nucleic acid sequence is ribozyme.

13. The method according to claim 9, further characterized in that said sequence of β-ketoacyl-ACP-synthase I is obtained from the endogenous host plant.

14. The method according to claim 13, further characterized in that said endogenous host plant is selected from the group consisting of Brassica, soybeans and corn.

15. The method according to claim 9, further characterized in that said host plant cell is a seed cell.

16. The method according to claim 9, further characterized in that said polyunsaturated fatty acid composition of the host plant cell is less than 13 mole percent.

17. An oil obtained from a plant cell produced by the method according to claim 1 and 9.

18. - A seed oil according to claim 9, further characterized in that it is obtained from a Brassica plant.

19. An isolated DNA sequence that encodes a soy protein β-ketoacyl-ACP synthase I.

20. The coding DNA sequence according to claim 19, further characterized in that said β-ketoacyl-ACP synthase I protein is encoded by a sequence comprising an EST selected from the group consisting of SEQ ID NOs: 2-5.

21. An isolated DNA sequence that encodes a soy protein β-ketoacyl-ACP synthase II.

22. The coding DNA sequence according to claim 21, further characterized in that said β-ketoacyl-ACP synthase II protein is encoded by a sequence comprising an EST selected from the group consisting of SEQ ID NOs: 6- 14

23. A method for increasing the medium chain fatty acid composition of a host plant cell, comprising growing a plant that has; a first introduced nucleic acid construct, said construct comprised in the 5 'to 3' direction of transcription, a functional promoter in a host plant cell, at least a portion of a nucleic acid sequence encoding a β-ketoacyl-protein ACP synthase I, and a region determining functional transcription in a plant cell, and a second introduced construct comprised in the 5 'to 3' direction of transcription, a functional promoter in a host plant cell, a nucleic acid sequence that encodes a preferred medium chain acyl-ACP thioesterase, and a functional transcription determination region in a plant cell.

24. The method according to claim 23, further characterized in that said nucleic acid sequence of said first construct is in an antisense orientation with respect to said promoter.

25. The method according to claim 23, further characterized in that said nucleic acid sequence of said first construction is in a sense orientation with respect to said promoter.

26. The method according to claim 23, further characterized in that said nucleic acid sequence of said first construct is a ribozyme.

27. The method according to claim 23, further characterized in that said sequence of β-ketoacyl-ACP synthase I is obtained from the endogenous host plant.

28. The method according to claim 27, further characterized in that the endogenous host plant is selected from the group consisting of: Brassica, soybeans and corn.

29. The method according to claim 23, further characterized in that said host plant cell is a seed cell.

30. - An oil obtained from a plant cell produced by the method according to claim 23.