CA2000661C - A process for the production of transgenic plants with increased nutritional value via the expression of modified 2s storage albumins - Google Patents

Abstract

The invention pertains to a process for producing transgenic plants with increased nutritional value. It comprises :
- cultivating plants obtained from regenerated plant cells or from seeds of plants obtained from said regenerated plant cells over one or several generations, whose genetic patrimony, replicable with said plants, comprises a precursor-coding nucleic acid sequence encoding the precursor of a 2S albumin storage protein and placed under the control of a promoter capable of directing gene expression in plants, said precursor-coding nucleic acid being modified in a nonessential region of its relevant sequence which encodes the mature 2S albumin or a subunit thereof with a nucleic acid insert in appropriate reading frame relationship with the surrounding part of said relevant sequence, said insert including a determined segment encoding an heterologous determined polypeptide containing appropriate aminoacid such as lysine and/or methionine and/or threonine and/or phenylalanine and/or tryptophane and/or leucine and/or valine and/or isoleucine.

Description

20~06~;~
A 1 2 j 1 9 97 A process for the production of transgenic plants with increased nutritional value via the expression of modified 2S storage albumins This invention relates to a process for the production of plants with increased content of appropriate aminoacids having high nutritional properties through the modification of plant genes encoding plant storage proteins, more particu-larly the 2S albumins.
More particularly, the invention aims at providing genetically modified plant DNA and plant live material in-cluding said genetically modified DNA replicable with the cells of said plant material, which genetically modified plant DNA contains sequences encoding for a polypeptide containing said appropriate aminoacids which expression is under the control of a suitable plant promoter.
A further object of the invention is to take advantage of the capacity of 2S albumins to be produced in large amounts in plants.
A further object of the invention is to take advantage of a hypervariable region of the 2S albumins, which supple-mentation with a number of said appropriate aminoacid codons in said hypervariable region of the gene enco~ing said 2S
albumins, do not disturb the correct expression! processing and transport of said produced modified storage proteins in the protein bodies of the plants.
Animals and men obtain directly or indirectly their essential aminoacids by eating plants. These essential aminoacids include lysine, thryptophane, threonine, methion-ine, phenylalanine, leucine, valine and isoleucine. For the easiness of the language these aminoacids are called "appro-priate aminoacids". Rather recently, agricultural scien-tists concerned with the world's hungry problem, concentrat-ed their work on developing plants with high nutritional yield. These new varieties, obtained through breeding in OGC, ~~

the most cases, were richer in carbohydrates but usually poorer in essential proteins than the wild type varieties from which they were derived. Currently, increasing recogni-tion of the role of plants in supplying essential aminoacids to the animal world had led to emphasis on the development of new food plants having a better aminoacid content.
Classical breeding however has limitations for achieving this goal. Molecular genetics, on the contrary, offers a possibility to overcome these difficulties. Reference is lo made to EP 0 208 418 published January 14, 1987 and the communication of Brown et al., 1986, in which a gene encod-ing a corn seed storage protein, (the so called zeins) is modified by the addition of sequences encoding lysine codons.
Seed storage proteins represent up to 90% of total seed protein in seeds of many plants. They are used as a source of nutrition for young seedlings in the period immediately after germination. The genes encoding them are strictly regulated, being expressed in a highly tissue specific and stage specific fashion (Walling et al., 1986; Higgins, 1984). Thus they are expressed almost exclusively in devel-oping seed, and different classes of seed storage proteins may be expressed at different stages in the development of the seed. They are generally restricted in their intercellu-lar location, being stored in membrane bound organelles called protein bodies or protein storage vacuoles. These organelles provide a protease-free environment, and often also contain protease inhibitors. A related group of pro-teins, the vegetative storage proteins, have similar ami-noacid compositions and are also stored in specialized vac-uoles, but are found in leaves instead of in seeds (Staswick, 1988). These proteins are degraded upon flower-ing, and are thought to serve as a nutritive source for developing seeds.

The expression of foreign genes in plants is well estab-lished (De Blaere et al., 1987). In several cases seed stor-age protein genes have been transferred to other plants. In most of these cases it was shown that within its new environ-ment the transferred seed storage protein gene is expressed in a tissue specific and developmentally regulated manner (Beachy et al., 1985; Sengupta-Gopalan et al., 1985; Marris et al., 1988; Ellis et al., 1988; Higgins et al., 1986, Oka-muro et al., 1986). It has also been shown in at least two cases that foreign seed storage proteins are located in the protein bodies of the host plant (Greenwood and Chrispeels, 1985;
Hoffman et al., 1987). It has further been shown that stable and functional messenger RNA's can be obtained if a cDNA, rather than a complete gene including introns, is used as the basis for the chimeric gene (Chee et al., 1986).
Storage proteins are generally classified on the basis of solubility and size (more specifically sedimentation rate, for instance as defined by Svedberg (in Stryer, L., Biochemistry, 2nd ed., W.H. Freeman, New York, page 599)). A
particular class of seed storage proteins has been studied, the 2S seed storage proteins, which are water soluble albu-mins. They represent a significant proportion of the seed storage proteins in many plants (Youle and Huang, 1981) (Table I) and their small size and consequently simpler structu~e makes t~em an attractive target for modification (see also EP 0 319 353 published June 7, 1989) . Several 2S
storage proteins have been characterized at either the pro-tein, cDNA or genomic clone levels (Crouch et al., 1983;
Sharief and Li, 1982; Ampe et al., 1986; Altenbach et al., 1987; Ericson et al., 1986; De Castro et al., 1987; Scofield and Crouch, 1987; Josefsson et al., 1987; EP 0 319 353, Krebbers et al., 1988). 2S albumins are formed in the cell from two subunits of 6-9 and 3-4 kilodaltons (kd) respective-ly, which are linked by disulfide bridges.

-2 ~
4 M A I 2 ~ 1~ 97 The work in the references above showed that 2S albu-mins are synthesized as complex prepropeptides whose organi-zation is shared between the 2S albumins of many different species and are shown diagrammatically for three of these species in figure 1. Several complete sequences are shown in figure 2.
As to Fig. 2 relative to protein sequences of 2S albu-mins, the following observations are made. For B. napus, ~.
excelsia, and A. thaliana both the protein and DNA sequences have been determined, for R. communis only the protein se-quence is available (B. napus from Crouch et al., 1983 and Ericson et al., 1986; B. excelsia from Ampe et al., 1986, De Castro et al., 1987 and Altenbach et al., 1987, _. communis from Sharief and Li, 1982). Boxes indicate homologies, and raised dots the position of the cysteines.
Comparison of the protein sequences at the beginning of the precursor with standard consensus sequences for signal peptides reveals that the precursor has not one but two segments at the amino terminus which are not present in the mature protein, the first of which is a signal sequence (Perlman and Halvorson, 1983) and the second of which has been designated as the amino terminal processed fragment (the so-called ATPF). Signal sequences serve to ensure the co-translational transport of the nAsc~nt polypeptide across the membrane of the endoplasmic reticulum (Blobel, 1980), and are found in many types of proteins, including all seed storage proteins examined to date (Herman et al., 1986). This is crucial for the appropriate compartmentalization of the pro-tein. The protein is further folded in such a way that cor-rect disulfide bridges are formed. This process is probably localized at the luminal site of the endoplasmatic reticulum membrane, where the enzyme disulfide isomerase is localized (Roden et al., 1982; Bergman and Kuehl, 1979). After translo-cation across the endoplasmic reticulum membrane it is thought that most storage proteins are transported via said (~iGC, TECI~ISO~IRCE

2 ¦ 19~7 endoplasmic reticulum to the Golgi bodies, and from the lat-ter in small membrane bound vesicles (~dense vesiclesn) to the protein bodies (Chrispeels, 1983; Craig and Goodchild, 1984; Lord, 1985). That the signal peptide is removed co-translationally implies that the signals directing the fur-ther transport of seed storage proteins to the protein bodies must reside in the remainder of the protein sequence present. Zeins and perhaps some other prolaminins deviate from this pathway; indeed the protein bodies are formed by budding directly off of the endoplasmic reticulum (Larkins and Hurkman, 1918). As already of record, 2S albumins contain sequences at the amino end of the precursor other than the signal sequence which are not present in the mature polypep-tide. This is not general to all storage proteins. This amino terminal processed fragment is labeled ATPF in figure 1.
In addition, as shown in figure 1, several aminoacids located between the small and large subunits in the precursor are removed (labeled IPF in the figure, which stands for internal process~ fragment). Furthermore, several residues are removed from the carboxyl end of the precursor (labeled CTPF in the figure which stands for carboxyl terminal pro-cessed fragment). The cellular location of these latter pro-cessing steps is uncertain, but is most likely the protein bodies (Chrispeels et al., 1983; Lord, 1985). As a result of these processing steps the small subunit and the large sub-unit remain. These are linked by disulfide bridges, as dis-cussed below.

When the protein sequences of 2S albumins of different plants are compared strong structural similarities are ob-served. This is more particularly illustrated by figure 2 which provides the aminoacid sequences of the small subunit and large subunit respectively of representative 2S storage seed albumin proteins of different plants, i.e.,:
R. comm. : Ricinus communis GC T~c~o~ C~

0 fi ~ ~ ~ A i 2 ¦ 19 ~ 7 A. thali.: Arabidopsis thaliana B. napus : Brassica napus B. excel.: Bertholletia excelsia (Brazil nut) It must be noted that in Fig. 2:
- the aminoacid sequences of said subunits extend on several lines; the cysteine y~OU~_ of the aminoacid sequences of the exemplified storage proteins and iden-tical aminoacids in several of said proteins have been brought into vertical alignment; the hyphen signs which appear in some of these sequences represent absent aminoacids, in other words direct linkages between the closest aminoacids which surrounded them:
- the aminoacid sequences which in the different proteins are conserved are framed.
It will be observed that all the sequences contain eight cysteine residues (the first and second in the small subunit, the remainder in the large subunit) which could participate in disulfide bridges as diagrammatically shown in Fig. 3, which represents a hypothetical model (for the purpose of the present discussion) rather than a representa-tion of the true structure of the 2S albumin of Arabidopsis thaliana.
Said hypothetical model has been inspired by the dis-ulfide ~ridge mediated loop-formation of animal albumins, such a~ serum albumins (Brown, 1976), alpha-fetoprotein (Jagodzinski et al., 1987; Morinaga et al., 1983) and the vitamine D binding protein where analogous constant C-C
doublets and C-X-C triplets were observed (Yang et al., 1985).
As can be seen on Fig. 2, the regions which are interca-lated between the first and second cysteines, between the fifth and sixth cysteines, and between the seventh and eight cysteines of the mature protein show a substantial degree of conservation or similarity. It would thus seem that these regions are in some way essential for the proper folding OGC, TECHSol JR~'J~

6 ~ ~, 2¦! t997 and/or stability of the protein when synthesized in the plants. An exception to this conservation consist in the distance between the sixth and seventh cysteine residues. This suggests that these arrangements are structurally important, but that some variation is permissi-ble in the large subunit between said sixth and seventh cys-teines where little conservation of aminoacids is observed.
An analogous suggestion has been made by Slightom and Chee (1987), where the viciline type seed storage proteins from peas were compared. These authors indeed suggest that ami-noacid replacement mutations designed to increase the number of sulphur containing aminoacids should be placed in regions which show little or no conservation of aminoacid sequences.
The authors however conclude that the proof that such modifi-cations can be tolerated will need to be tested in the seeds of transgenic plants. Moreover, the teaching provided in their paper on the properties of the through deletion modi-fied storage protein concerns only the influence on expres-sion levels and not on processing of said storage proteins.
An embodiment of this invention is the demonstration that a well chosen region of the 2S albuuin allows variation without altering the properties and correct processing of said modified storage protein in plant cells of transgenic plants.
This region (diagrammatically shown in Fig. 3 by an enlarged hatched portion) will in the examples hereafter referred to be termed as the "hypervariable region~. Fig. 3 also shows the respective positions of the other parts of the precursor sequence, including the "IPF~ section separating the small subunit and large subunit of the precursor, as well as the number of aminoacids (aa) in substantially conserved portions of the protein subunits cysteine residues. The pro-cessing cleavage sites (as determined by Krebbers et al., 1988) are shown by symbols.

OGC, T~CHSf~

... ~Q~6~ Y2~;'1997 The seeds of many plants contain albumins of approximate-ly the same size as the storage proteins discussed above.
However, for ease of language, this document will use the term "2S albumins" to refer to seed proteins whose genes encode a peptide precursor with the general organization shown in figure 1 and which are processe~ to a final form consisting of two subunits linked by disulfide bridges. The process of the invention for producing plants with an in-creased content of appropriate aminoacids comprises :
cultivating plants obtained from regenerated plant cells or from seeds of plants obtained from said regenerated plant cells over one or several generations, wherein the genetic patrimony or information of said plant cells, replicable within said plants, includes a nucleic acid sequence, placed under the control of a plant promoter, which can be transcribed into the mRNA encoding at least part of the precursor of a 2S albumin including the signal peptide of said plant, said nucleic acid being hereafter referred to as the "precursor encoding nucleic acid"
. wherein said nucleic acid contains a nucleotide se-quence (hereafter termed the "relevant sequencen) which relevant sequence comprises a nonessential region modi-fied by a heterologous nucleic acid insert forming an open reading frame in reading phase with the non modi-fied parts surrounding said insert in said relevant sequence.
. wherein said insert includes a nucleotide segment encoAing a polypeptide containing appropriate ami-noacids.
It will be appreciated that under the above mentioned conditions each and every cell of the cultivated plant will include the modified nucleic acid. Yet the above defined recombinant or hybrid sequence will be expressed at high levels constitutively or only or mostly in certain organs of QGC TECH.SrJt I~CE

~ ~ ~ Q ~fi ~ ~Y 21 ~997 the cultivated plantc dependent on which plant promoter ha~
been chosen to conduct its expression. In the case of seed-specific promoters the hybrid storage protein will be produced mostly in the seeds.
It will be understood that the ~heterologous nucleic acid insert" defined above consists of an insert which con-tains nucleotide sequences which at least in part, may be foreign to the natural nucleic acid encoding the precursor of the 2S albumins of the plant cells concerned and encode the appropriate aminoacids. Most generally the segment encoding polypeptide containing said appropriate aminoacids will it-self be foreign to the natural nucleic acid enco~;nq the precursor of said storage protein. Nonetheless, the ter~
"heterologous nucleic acid insertN does also extend to an insert containing a segment as above-defined normally present in the genetic patrimony or information of said plant cells, the "heterologous" character of said insert then addressing to the different genetic environment ~hich surrounds said insert.
In the pr~ceAing definition of the process according to the invention the so-called "nonessential region~ of the relevant sequence of said nucleic acid encoding the precur-sor, consists of a region whose nucleotide sequence can be modified either by insertion into it of the above-defined insert or by replacement of at least part of said nonessen-tial region by said insert, yet without disturbing the stabil-ity and correct processing of said hybrid storage protein a~
well as its transport into the above-said protein bodies.
Sequences consisting of said insert or replacement and repre-senting the coding region for a polypeptide containing appro-priate aminoacids can either be put in as synthetic oligomers or as restriction fragments isolated from other genes, as thought by Brown, 1986. The total length of the hybrid stor-age protein may be longer or shorter than the total length of the non-modified 2S albumin.

~)GC, TECHSOURC~

With respect to the choice of the region to be modified, the present invention is clearly distinguishable from other work which has been done in this field. Reference is made to DD-A-240911 patent from the Akademie der Wissenschaften der DDR where legumin genes from Vicia faba, (glutine and prola-mine) were modified in vitro with sequences encoding methion-ine. As place of insert a natural occurring PstI site has been chosen. At the EMBO workshop "Plant storage protein genes", (Breisach, FRG, September 1986) the authors presented their work and informed the audience that plant transforma-tion experiments were ~ust started with the modified gene.
Mo further results have yet ~eer, published.
Reference is also made to patent application WO 87/07299 published December 3, 1987 and correspon~;ng publication of Radke et al., 1988. These papers describe the modification of the napin gene, which encodes the 2S albumin o~ Brassica napus, ~y a r.ucleotide sequence encoding nine aminoacid residues includ-ing 5 consecutive methionines. The region of modification is a naturally occurring SstI site within the region encoding the mature protein. Such a modification would result in a insertion directly adjacent to a cysteine residue and more-over in a region between two cysteines, namely the 4th and the 5th cysteines of the mature protein which correspond with the 2nd and 3rd cysteines of the large subunit, whose length is strongly conserved (see above). We believe such a modifi-cation is likely to disrupt a normal folding and stability of the 2S albumin (EP 0 319 353 publiRhed June 7, 1989). Moreover, above cited reference~ provide no evidence that the desired modi-fied 2S albumin was successfully synthesized, correctly pro-cessed or correctly targeted.
In the present invention the precursor-coding nucleic acid referred to above may of course originate from the same plant species as that which is cultivated for the purpose of the invention. It may however originate from another plant species, in line with the teachings of Beachey et al., 1985 and Okamurc et al., 1986 already of record.

2 0 ~ ~ 6 ~ Y 21-j 19~7 In a similar manner the plant promoter may originate from the same plant ~pecies or from another, sub~ect in the last instance to the capability of the host plant's polymeras-es to reco~nize it. It may act constitutively or in a tissue-specific manner, such as, but not limited to, seed-specific promoters.
Regions such as the ones at the end of the small sub-unit, at the beginning or end of the large subunit, show differences of such a magnitude that they can be held as presumably having no substantial impact on the final proper-ties of the protein. The extreme carboxyl terminus of the small subunits and the amino terminus of the large subunit may, however, be involved in the processing of the internal proceC~s~ fragment. A region which does not seem essential, consists of the middle position of the region located in the large subunit, between the sixth and the seventh cysteine of the nature protein, but not immediately adjacent and at least 3 aminoacids separated from said cysteines. Thus in addition to the absence of similarity at the level of the aminoacid residues, there appears a difference in length which makes that region eligible for substitutions in the longest 2S
albumins and for addition of aminoacids in the shortest 2S
albumins or for elongation of both. The same should be appli-cable at approximately of the end of the first third part of the same region between said sixth and seventh cysteine; see the sequence of R. communis which is much shorter at that region than the corresponding regions of the other exempli-fied 2S proteins.
It is of course realized that caution must be exorcised against hypotheses based on arbitrary choices as concerns the bringing into line of similar parts of proteins which else-where exhibit substantial differences. Nevertheless such comparisons have proven in other domains of genetics to pro-vide the man skilled in the art with appropriate guidance to reasonably infer from local structural differences, on the C)GC, T~CL~S~,~J~E

2 ~ 6 ~ 21 19~7 one hand, and from local similarities, on the othe~ hand, in similar proteins of different sources, which parts of such proteins can be modified and which parts cannot, when it is sought to preserve some basic properties of the non modified protein in the same protein yet locally modified by a foreign or heterologous sequence.
The choice of the adequate nonessential regions to be used in the process of the invention will also depend on the length of the polypeptide containing the appropriate ami-noacids. Basically the method of the invention allows the modification of said 2S albumins by the insertion and/or partial substitution into the precursor nucleic acid of se-quences encoding up to 100 aminoacids.
When the complete protein sequence of the region to be inserted into a 2S albumin has been determined, the nucleo-tide sequence to encode said protein sequence must be deter-mined. It will be recognized that while perhaps not absolute-ly neces~Ary the codon usage of the encoAing nucleic acid should where possible be similar to that of the gene being modified.
The person skilled in the art will have access to appropriate computer analysis tools to determine said codon usage.
Any appropriate genetic engineering tec~nique may be used for substituting the insert for part of the selected precursor-coding nucleic acid or for inserting it in the appropriate region of said precursor-coA i ng nucleic acid. The general n vitro recombination techniques followed by cloning in bacteria can be used for making the chimeric genes.
Site-directed mutagenesis can be used for the same pU~ 5 as further exemplified hereafter. DNA recombinants, e.g.
plasmids suitable for the transformation of plant cells can also be produced according to techniques disclosed in current technical literature. The same applies finally to the produc-tion of transformed plant cells in which the hybrid storage protein encoded by the relevant parts of the selected O~JC, T~CH.~~,' J~Cf~

6 ~ ~
~Y 21 1997 precursor-coding nucleic acid can be expressed. By way of example, reference can be made to the published European applications no. 116 718 or to International application WO
84/02913 and, which disclose appropriate te~hniques to that effect.
When designing the sequences rich in appropriate ami-noacids, care must be taken that the resulting peptide con-taining said appropriate aminoacids does not influence the stability of the modified 2S albumin. Certain insertions may indeed disrupt the structure of the protein. For example, long stretches of methionines may result in rod ~hAp~d heli-ces which would result in instabilities due to disruption of normal folding patterns. Thus such sequences must occasional-ly include aminoacids which interrupt the helical structure.

The procedures which have been disclosed hereabove apply to the adequate modification of the nonessential region of any of 2S albumins by an heterologous insert containing a DNA
sequence enco~ing a peptide containing appropriate aminoacids with nutritional properties and then to the transformation of the relevant plants with the chimeric gene obtained for the production of a hybrid protein containing the Fe~nce of said peptide in the cells of the relevant plant. Needless to say that the person skilled in the art will in all ins~nc~C be able of selecting which of the existing tec~ique~ would at best fulfill its needs at the level of each step of the produc-tion of such modified plants, to achieve the best production yields of said hybrid storage protein.
For instance the following process can be used in order to exploit the capacity of a 2S albumin, to be used as a suit-able vector for the production of plants with increased nutri-tional value, by inserting in said 2S albumins nucleotide codons encoding methionine and/or lysine and/or thryptophane and/or threonine and/or phenylalanine and/or leucine and/or valine and/or isoleucine when the corresponding OGC, TEC~Is~! J~rr 6 fi ~ 21 1997 precursor-coding nucleic acid has been sequenced. Such process then comprises:
1) locating and selecting one of said relevant sequences of the precursor-coding nucleic acid which comprises a nonessential region enco~ing a peptide sequence which can be modified by substituting an insert for part of it or by inserting of said insert into it, which modification is compatible with the conservation of the configuration of said 2S albumins and this preferable by determining the relative positions of the codons which encode the successive cysteine residues in the mature protein or protein subunits of said 2S albumins and identifying the corresponding successive nucleic acid regions located upstream of, between, and downstream of said codons with-in said sub-sequences of the precursor-coding nucleic acid and identifying in said successive regions those parts which undergo variability in either aminoacid se-quence or length or both from one plant species to anoth-er as compared with those other regions which do exhibit substantial conservation of aminoacid sequence in said several plant species, one of said nucleotide regions being then selected for the insertion therein of the nucleic acid insert as described hereunder.
An alternative would consist of st,udying any 3-D struc-tures which may become available in the future.

2)inserting a nucleic acid insert in the selected region of said precursor nucleic acid in appropriate reading frame relationship with the non-modified parts of said relevant sequence, which insert includes a determined segment enco~;~g a peptide containing all or part of the above mentioned appropriate aminoacids.
3) inserting the modified precursor-coding nucleic acid obtained in a plasmid suitable for the transformation of plant cells which can be regenerated into full OGC, T~C~ r~

~l 21 ~g97 seed-forming plants, wherein ~aid insertion is brought under the control of regulation elements, particularly a plant pro~oter capable of providing for the exp~ ion of the open reading-frames associated therewith in said plants;

4) transforming a culture of such plant cells with such modified plasmid;

5) assaying the expression of the chimeric gene encoding the hybrid storage protein and, when achieved;

6) regenerating said plants from the transformed plant cells obtained and growing said plants up to maturity.
In the case the chimeric gene is under the control of a seed specific promotor, growing up the transformed plants to seeds must precede step 5) Hence embodiment as described under 1) of the invention hereabove provides that in having the hybrid 2S albumins in a plant, it will pass the plant protein disulfide isomerase during membrane translocation, thus increasing the chances that the correct disulfide bridges be formed in the hybrid precursor as in its normal precursor situation, on the one hand The invention further relates to the recombinant nucleic acids themselves for use in the process of the invention;
particularly to the - recombinant precursor encoding nucleic acid defined in the context of said process;
- recombinant nucleic acids containing said modified precursor encoding nucleic acid under the control of a plant promoter, whether the latter originates from the same DNA as that of said precursor coding nucleic acid or from another DNA of the same plant from which the precursor encoding nucleic acid is derived, or from a DNA of another plant, or from a non-plant organism provided that it is capable of directing gene expression in plants.

OGC TEC!~ rF

fi ~
~ 9 9 7 - vectors, more particularly plant plasmids e.g., Ti-derived plasmids modified by any of the preceding recombinant nucleic acids for use in the transforma-tion of the above plant cells.
The invention also relates to the regenerable source of the hybrid 2S albumin, which is formed of in the cells of a seed-forming-plant, which plant cells are capable of being regenerated into the full plant or seeds of said seed-forming plants wherein said plants or seeds have been obtained as a result of one or several generations of the plants resulting from the regeneration of said plant cells, wherein further the DNA supporting the genetic information of said plant cells or seeds comprises a nucleic acid or part thereof, including the sequences encoding the signal peptide, which can be transcribed in the mRNA corresponding to the precursor of a 2S albumin of said plant, placed under the control of a plant specific promoter, and . wherein said nucleic acid sequence contains a relevant modified sequence encoding the mature 2S storage protein or one of the several sub-sequences encoding for the corresponding one or several sub-units of said mature 2S
albumins, . wherein further the modification of said relevant sequence takes place in one of its noneccential regions and consists of a heterologous nucleic acid insert form-ing an open-reading frame in reading phase with non modified parts which surround said insert in the rele-vant sequence, . wherein said insert consists of a nucleotide segment e~coAing a peptide containing methionine and/or lysine and/or thryptophane and/or threonine and/or phenylala-nine, and/or leucine and/or valine and/or isoleucine.
It is to be considered that although the invention should not be deemed as being limited thereto, the nucleic inserts encoding the above mentioned appropriate aminoacids QGC TEC~Sf',~ F

fi ~ y 2~ ly97 will in most instances be man-made synthetic oligonucleotides or oligonucleotides derived from procaryotic or eucaryotic genes or of from cDNAs derived of procaryotic or eucaryotic RNAs, all of which shall normally escape any possibility of being inserted at the appropriate places of the plant cells or seeds of this invention through biological proceC~?-~
whatever the nature thereof. In other vords, these inserts are "non plant variety specific~, specially in that they can be inserted in different kinds of plants which are genetical-ly totally unrelated and thus incapable of eY~h~nging any genetic material by standard biological procesces, including natural hybridization process~s.
Thus the invention further relates to the seed forming plants themselves which have been obtained from said trans-formed plant cells or seeds, which plants are characterized in that they carry said hybrid precursor-coding nucleic acids associated with a plant promoter in their cells, said inserts however being expressed and the corresponding hybrid protein produced in the cells of said plants.
There follows an outline of a preferred method which can be used for the modification of a 2S albumin gene and its expression in the seeds obtained from the transgenic plants.
The outline of the method given here is fol~owed by a specif-ic example. It will be understood from the person skilled in the art that the method can be suitably adapted for the modi-fication of other 2S albumin genes.
1. Replacement or supplementation of the hypervariable region of the 2S albumin gene by a sequence encoding peptide containing appropriate aminoacids which possess nutritional properties.
Either the cDNA or the genomic clone of the 2S albumin can be used. Comparison of the sequences of the hypervariable regions of the genes in figure 2 shows that they vary in length. Therefore if the sequence encoding a peptide contain-OGC, TrCHSCUF~CE

ing the appropriate aminoacids is short and a 2S albumin with a relatively short hypervariable region is used, said se-quence of interest can be inserted. Otherwise part of the hypervariable region is removed, to be replaced by the insert containing a larger segment or sequence encoding the peptide containing the appropriate aminoacids. In either case the modified hybrid 2S albumin may be longer than the native one. In either case two standard techniques can be applied;
convenient restriction sites can be exploited, or mutagenesis vectors (e.g. Stanssens et al. 1987) can be used. In both cases, care must be taken to maintain the reading frame of the message.
The sequence encoding the signal peptide of the precur-sor of the storage protein used either belongs to this precur-sor or can be a substitute sequence coding for the signal peptide or peptides of an heterologous storage protein.
2. The altered 2S albumin coding region is placed under the control of a plant promoter. Preferred promoters in-clude the strong constitutive exogeneous plant promoters such as the promoter from cauliflower mozaic virus di-recting the 35S transcript (Odell, J.T. et al., 1985), also called the 35S promoter; the 35S promoter from the CAMV isolate Cabb-JI (Hull and Howell, 1987), also called the 35S3 promoter; the bidirectional TR promoter which drives the expression of both the 1' and the 2' genes of the T-DNA (Velten et al., 1984).
Alternatively a promoter can be utilized which is not constitutive but specific for one or more tissues or organs of the plant. Given by way of example such kind promoters may be the light inducible promoter of the ribulose-l, 5-bi-phosphate carboxylase small subunit gene (EP 0 193 259 published December 3, 1986, if the expres-sion is desired in tissue with photosynthetic activity, or may be seed specific promoters.

19 ~ 6 ~ ~a~r 21 A seed ~pecific promoter is used in order to ensure subsequent expression in the seeds only. This may be of particular use, since ~eeds constitute an important food or feed source. Moreover, this specific expression avoids possi-ble stresses on other parts of the plant. In principle the promoter of the modified 2S albumin can be used. But this is not necessAry. Any other promoter serving the same purpose can be used. The promoter may be chosen according to its level of efficiency in the plant species to be transformed.
In the examples below the 2S albumin promoter from the 2S
albumin gene from Arabidopsis is used, which constitutes the natural promotor of the 2S albumin gene which is modified in said examples. Needless to say that other seed specific promo-tors may be used, such as the conglycinine promotor from soybean. If a chimeric gene is so constructed, a signal pep-tide encoAin~ region must also be included, either from the modified gene or from the gene whose promotor is being used.
The actual construction of the chimeric gene is done using stAn~rd molecular biological tech~ques described in Mania-tis et al., 1982. (see example).
3. The chimeric gene construction is transferred into the appropriate host plant.
When the chimeric or modified gene construction is com-plete it is transferred in its entirety to a plant transforma-tion vector. A wide variety of these, based on disarmed (non-oncogenic) Ti-plasmids derived from A~robacterium tumefa-ciens, are available, both of the binary and cointegration forms (De Blaere et al., 1987). A vector including a selectable marker for transformation, usually antibiotic resistance, should be chosen. Similarly, the methods of plant transformation are also numerous, and are fitted to the individual plant. Most are based on either protoplast transformation (Marton et al., 1979) or formation of a small piece of tissue from the adult plant (Horsch et al., 1985).
In the example below, the vector is a binary disarmed C)GC TECH~ l lRC~

~3Q~
I'Vl A 1 2 l ~997 Ti-plasmid vector, the marker is kanamycin resistance, and the leaf disc method of transformation is used.
Calli from the transformation procedure are selected on the basis of the selectable marker and regenerated to adult 5plants by appropriate hormone induction. This again varie~
with the plant species being used. Regenerated plants are then used to set up a stable line from vhich seeds can be harvested.
Further characteristics of the invention will appear in 10the course of the non-limiting disclosure of specific exam-ples, particularly on the basis of the drawings in which:

- Figs. 1, 2 and 3 refer to overall features of 2S-albumins as already discussed above. The numbers 15refer to the number of aminoacids observed in the different fragments of the protein precursor.
- Fig. 4 represents the sequence of lkb fragment con-taining the Arabidopsis thaliana 2S albumin gene and shows related elements. The NdeI site is underlined.
20- Fig. 5 provides the protein sequence of the large subunit of the above Arabidopsis 2S protein together with related oligonucleotide sequences.
- Fig. 6A shows diagrammatically the successive phA~eF
of the construction of a chimeric 2S albumin-Arabidop-25thaliania gene including the deletion of practi-cally all parts of the hypervariable region and its replacement by a AccI site, the insertion of DNA
sequences rich in methionine codons, given by way of of example in the following disclosure, in the AccI
30site, particularly through site-directed mutagenesis and the cloning of said chimeric gene in plant vector suitable for plant transformation.
- Fig. 6B shows diagrammatically t~e protein sequence of the large subunit of several Arabidopsis 2S albu-35mins and indicates the region removed from the genes ~G~ TEC~S r!!!~ ~ F

2 (~ ~ n 6 ~ 11 M,~Y 21 1~97 enco~;ng said 2S albumins, and shows diagrammatic~lly where an AccI site has been created and how oligonu-cleotides rich in methionine codons are inserted into said AccI site in such a way that the open re~ng frame is maintained.

- Fig 7 diagrammatically compares the protein sequenc-es of the large subunits of the unmodified 2S albu-min, in which most of the hypervariable region has been deleted, and those of the modified 2S albu-mins. The resulting number of methionine residues are indicated.
- Fig. 8 shows the restriction sites and genetic map of a plasmid suitable for the performance of the above site-directed mutagenesis.
- Fig. 9 shows diagrammatically the different steps of the site-directed mutagenesis procedure of St~nc~Qn~
et al (1987) as generally applicable to the modifica-tion of nucleic acid at appropriate places.
- Fig. 10 gives the restriction map of pGSC1703A.
Example I :
As a first example of the method described, a prGc~
is given for the production of transgenic plant seeds with increased nutritional value by having inserted into tbeir genome a modified 2S albumin protein from Arabidopsis thaliana having deleted its hypervariable region and re-placed by way of example by a methionine rich peptide hav-ing 7 aminoacids with the following sequence :I M M M M R
M. A synthetic oligomer encoAing said peptide is substitut-ed for essentially the entire part of the hypervariable region in a genomic clone encoding the 2S albumin of Arabi-dopsis thaliana. Only a few aminoacids adjacent to the sixth and seventh cysteine residues remained. This chimer-ic gene is under the control of its natural promoter and ;3GC, ~:C'~SClJRcE

6 ~ ~Y
~' 21 ~9~7 signal peptide. The process and constructions are diagram-matically illustrated in Fiq. 6A, 6B and 7. The entire construct is transferred to tobacco, Arabidopsis thalian~
and Brassica napus plants using an Agrobacterium mediated transformation system. Brassica napus is of particular interest, since this crop is widely used as protein source for animal feed.
Plants are regenerated, and after flowering the seeds are collected and the methionine content compared with untrans-formed plants.
1. Cloninq of the Arabidopsis thaliana 2S albumin gene.

The Arabidopsis thaliana gene has been cloned accord-ing to what is described in Krebbers et al., 1988. The plasmid containing said gene is called pAT2S1. The se-quence of the region containing the gene, which is called AT2Sl, is shown in figure 4.

2. Deletion of the hypervariable re~ion of AT2S1 gene and replacement by an AccI site.

Part of the hypervariable region of AT2S1 is replaced by the following oligonucleotide:

5'- CCA ACC TTG AAA GGT ATA CAC TTG CCC AAC - 3' 30-mer P T L K G I H L P N

in which the underlined sequences represent the AccI site and the surrounding ones sequences complementary to the cod-ing sequence of the hypervariable region of the Arabidopsis 2S albumin gene to be retained. This results finally in the aminoacid sequence indicated under the oligonucleotide.

O(~C, TF~-~HSCllR~E

The deletion and substitution of part of the sequence encod-ing the hypervariable region of AT2Sl is done using site directed mutagenesis with the oligonucleotide as primer. The system of Stanssens et al. (1987) is used.
The Stanssens et al. method is described in EP 0 319 353 publis-hed June 7, 1989. It makes use of plasmid pMac5-8 whose restriction and genetic m~Ap and the positions of the relevant genetic loci are shown in Fig. 8. The arrows denote their functional orientation.

origin of replication of filamentous phage fl; ORI:
ColE1-type origin of replication; BLA/ApR : region coding for B-lactamase; CAT/CmR : region coding for chlorampheni-col acetyl transferase. The positions of the amber mutations present in pMc5-8 (the bla-am gene does not contain the ScaI
site) and pMc5-8 (cat-am; the mutation eliminates the unique PvuII
site) are indicated. Suppression of the cat amber mutation in both suDE and supF hosts results in resistance to at least 25 ug/ml Cm. pMc5-8 confers resistance to +20 ug/ml and 100 ug/ml Ap upon amber-suppression in supE and supF strains respectively. The EcoRI, BalI and NcoI sites present in the wild-type cat gene (indicated with an asterisk) have been removed using mutagenesis techniques.
Essentially the mutagenesis round used for the above men-tioned substitution is ran as follows. Reference is made to Fig. 9, in which the amber mutations in the Ap and Cm select-able markers are shown by closed circles. The symbol represents the mutagenic oligonucleotide. The mutation itself is indicated by an arrowhead.

The individual steps of the process are as follows:

- Cloning of the HindIII fragment of pAT2Sl containing the coding region of the AT2Sl gene into pMaS-8 (I). This vector carries on amber mutation in the CmR gene and M ~ 1 21 1997 specifies resistance to ampicillin. The resulting plas-mid is designated pMacAT2S1 (see figure 6A step 1).
- Preparation of single stranded DNA of this recombinant (II) from pseudoviral particles.
- Preparation of a HindIII restriction fragment from the complementary pMc type plasmid (III). pMc-type vectors contain the wild type CmR gene while an amber mutation is incorporated in the Ap resistance marker.
- Construction of gap duplex DNA (hereinafter called gdDNA) gdDNA (IV) by in vitro DNA/DNA hybridization. In the gdDNA the target sequences are exposed as single stranded DNA. Preparative purification of the gdDNA from the other components of the hybridization mixture is not nececc~ry.
- Annealing of the 30-mer synthetic oligonucleotide to the gdDNA (V).
- Filling in the remaining single stranded gaps and seal-ing of the nicks by a simultaneous i~ vitro Klenow DNA
polymerase I / DNA ligase reaction (VI).
- Transformation of a mutS host, i.e., , a strain deficient in mismatch repair, selecting for Cm resis-tance. This results in production of a mixed plasmid progeny (VII).
- Elimination of progeny deriving from the template strand (pMa-type) by retransformation of a host unable to sup-press amber mutations (VIII). Selection for Cm resi~-tance results in enrichment of the progeny derived from the gapped strand, i.e., , the strand into which the mutagenic oligonucleotide has been incorporated.
- Screening of the clones resulting from the retransforma-tion for the presence of the desired mutation. The re-sulting plasmid containing the deleted hypervariable region of AT2Sl is called pMacAT2SlC40 (see figure 6A
step 2).

OGC, TECH~OURCF

6 ~ ~
~ 21 1997 3. Insertion of sequences rich in methionine codons into the AT2Sl gene whose sequences encoding the hypervariable region have been deleted.

As stated above when the sequences enco~;ng moct of the hypervariable loop were removed an AccI site was inserted in its place. The sequences of interest will be inserted into this AccI site, but a second AccI site is also present in the HindIII fragment containing the modified gene. Therefore the NdeI-HindIII fragment containing the modified gene is sub-cloned into the cloning vector pBR322 (Bolivar, 1977) also cut with NdeI and HindIII. The position of the NdeI site in the 2S albumin gene is indicated in figure 4. The resulting subclone is designated pBRAT2Sl (Figure 6A, step 3).

In principle any insert desired can be inserted into the AccI site in pBRAT2Sl. In the present example said insert encodes the following sequence: I.M.M.~.M.R.M. Therefore complementary oligonucleotides encoding said peptide are synthesized taking into account the codon usage of AT2Sl and ensuring the the ends of the two complementary oligonucleo-tides are complementary to the staggered ends of the AccI
site, as shown here (the oligonucleotides are shown in bold type) :

5' GT ATA AT& ATG AT& ATG CGC ATG ATAC 3' 3' CA TAT TAC TAC TAC TAC GCG TAC TATG 5' The details of this insertion, showing how the reading frame is maintained, are shown in figure 6B. The two oligonu-cleotides are annealed and ligated with pBRAT2Sl digested with AccI (figure 6A, step 4). The resulting plasmid is designated pAD4.

OGC, T~f~,'u.Sf.--;'. ''--, ~iA~ ~1 1997 4. Reconstruct~on of the com~lete modified AT2Sl gene with its natur~l ~romoter.

The complete chimeric gene is reconstructed a~ follows (see figure 6A): The clone pAT2SlBg contains a 3.6kb BglII
fragment inserted in the cloning vector pJB65 (Botterman et al., 1987) which encompasses not only the l.Okb HindIII frag-ment containing the coding region of the gene AT2Sl but suffi-cient sequences upstream and downstream of this fragment to contain all neceC~ry regulatory elements for the proper expression of the gene. This plasmid is cut with HindIII and the 5.2kb fragment (i.e., that portion of the plasmid not containing the coding region of AT2Sl) is isolated. The clone pAT2Sl is cut with HindIII and NdeI and the resulting 320 bp HindIII-NdeI fragment is isolated. This fragment represents the one removed from the modified 2S albumin in the construction of pBRAT2Sl (step 3 of figure 6A) in order to allow the insertion of the oligonucleotides in step 4 of figure 6A to proceed without the complications of an extra AccI site. These two isolated fragments are then ligated in a three way ligation with the NdeI-HindIII fragment from pAD4 (figure 6A, step 5) containing the modified co~3ing sequence.
Individual tranformants can be screened to check for appropri-ate orientation of the reconstructed HindIII fragment within the BglII fragment using any of a number of sites. The re-sulting plasmid, pAD17, consists of a 2S albumin gene modi-fied only in the hypervariable region, suLLo~"~ed by the same flanking sequences and thus the same promoter as the unmodi-fied gene, the entirety contained on a BglII fragment.

5. Transformation of plants.
The BglII fragment containing the chimeric gene is in-serted into the BglII site of the binary vector pGSC1703A
(Fig. 10) (see also Fig. 6A step 6). The resultant plasmid is designated pTAD12. Vector pGSC1703A contains functions OGC, TECHSGURCE

fi ~
~,A~ 21 ~997 for selection and stability in both ~. Çli and ~. tumefa-cien8, as well as a T-DNA fragment for the transfer of for-eign DNA into plant genomes (Deblaere et al., 1987). It fur-ther contains the bi-directional TR promotor (Velten et al., 1984) with the neomycin pho~photransferase protein coding region (neo) and the 3' end of the ocs gene on one side, and a hygromycin transferase gene on the other side, ~o that transformed plants are both kanamycin and hygromycin resis-tant. This plasmid does not carry an ampicillin resistance gene, so that carbenicillin as well as claforan can be used to kill Agrobacterium after the infection step. Using stan-dard procedures (Deblaere et al., 1987), pTAD12 is trans-ferred to the Agrobacterium strain C58ClRif carrying the plasmid pMP90 (Koncz and Schell, 1986). The latter provides in trans the vir gene functions required for successful trans-fer of the T-DNA region to the plant genome. This Agrobacteri-um is then used to transform plants. Tobacco plants of the strain SRl are transformed using st~n~rd procedures (De-blaere et al., 1987). Calli are selected on 100 ug/ml kan-amycin, and resistant calli used to regenerate plants.
The techniques for transformation of Arabidopsis thaliana and Brassica napus are such that exactly the same construc-tion, in the same vector, can be used. After mobilization to Agrobacterium tumefaciens as described hereabove, the proce-dures of Lloyd et al., (1986) and Rlimaszewska et al. (1985) are used for transformation of Arabidopsis and Brassica re-spectively. In each case, as for tobacco, calli can be se-lected on 100 ug/ml kanamycin, and resistant calli used to regenerate plants.
In the case of all three ~pecies at an early stage of regeneration the regenerants are checked for transformation by inducing callus from leaf on media supplemented with kan-amycin (see also point 6).

3 6. Screening and analysis of transformed plants.

6 ~ ~
28 I~'IAI 21 1997 In the cace of all three species, regenerated plants are grown to seed. Since different transformed plants can be expected to have varying levels of expression (~position effectsn, Jones et al., 1985), more than one tranformant must initially be analyzed. This can in principle be done at either the RNA or protein level; in this case seed RNA was prepared as described (Beachy et al., 1985) and northern blots carried out using standard tec-hniques (Thomas et al., 1980). Since in the case of both Brassica and Arabidopsis the use of the entire chimeric gene would result in cross hybridization with endogeneous genes, oligonucleotide probes complementary to the insertion within the 2S albumin were used; the same probe as used to make the construction can be used. For each species, 1 or 2 individual plants were chosen for further analysis as ~iscllcsed below.
First the copy number of the chimeric gene is determined by preparing DNA from leaf tissue of the transformed plants (Dellaporta et al., 1983) and probing with the oligonucleo-tide used above.
The methionine content of the seeds is analyzed using known methods (Joseph and Marsden, 1986; Gehrke et al., 1985;
~l~in and Griffith, 1985 (a) and (b)).

Example II

As a second example of the method described, the same procedure is followed for the production of transgenic plant seeds with increased nutritional value by having inserted into their genome a modified 2S albumin protein from ArabidoD-sis thaliana having deleted its hypervariable region and replaced by way of example by a methionine rich peptide hav-ing 24 aminoacids with the following sequence :

C ~C~' 29 M ~1 21 1997 I ~ ~ ~ Q P R G D ~ ~ Q P R G ~ ~ ~

All different steps going from constructs to transformants as disclosed for example I are executed with the only differ-ence that in step 3 the following oligonucleotide has been synthesized and inserted into pBrAT2Sl (the oligonucleotides are shown in bold type) 5 ' GT ATA ATG ATG ATG CAA CCA AGG GGC GAT ATG ATG ATG ATA
Aq~G ATG ATG
3' CA TAT TAC TAC TAC GTT GGT TCC CCG GTA TAC TAC TAC TAT
TAC TAC TAC

CAA CCA AGG GGC GAT ATG ATG ATG ATA C - 3' GTT GGT TCC CCG CTA TAC TAC TAC TAT G - 5 ' The relevant plasmids are indicated in figure 6A, details of the insertion in figure 6B and resulting aminoacid se-quence of the hybrid subunit shown in figure 7. The relevantplasmids as indicated in figure 6A are pAD3, pAD7 and pTAD10.

The examples have thus given a complete illustration of how 2S albumin storage proteins can be modified to incorpo-rate therein an insert enco~ing a methionine rich polypeptidefollowed by the transformation of plant cells such as tobacco cells, Arabidopsis cells and Brassica napus cells with an appropriate plasmid cont~in~ng the corresponding modified precursor nucleic acid, the regeneration of the transformed plant cells into correspon~ing plants, the culture thereof up to the seed forming stage, the recovery of the seeds and finally the analysis of the methionine content of said seeds compared with the seeds of corresponding non transformed plants.

It goes without saying that the invention is not limited to the above examples. The person skilled in the art will in each case be able to choose the desired combination of appro-priate aminoacids to be inserted into the hypervariable re-gion of the 2S storage protein, in function of the plant he wants to improve with regard to its nutritional value and in function of the desired application of the modified plant.
There follows a list of bibliographic references which have been referred to in the course of the present disclosure to the extent when reference has been made to known methods for achieving some of the process steps referred to herein or to general knowledge which has been established prior to the performance of this invention. -It is further confirmed - that plasmid pAT2Sl has been deposited with the DSM on 4879 on October 7, 1988 - plasmids pMa5-8 has been deposited with the DSM on 4567 and pMc on 4566 on May 3, 1988 - plasmid pAT2SlBg has been deposited with the DSM on 4878 on October 7, 1988 - plasmid pGSC1703a has been deposited with the DSM on 4880 on October 7, 1988 nowithstanding the fact that they all consist of constructs that the person skilled in the art can reproduce them from available genetic material without performing any inventive work.

3 ~ I MMAY 21 ~7 References :

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(1987) Plant Mol. Biol. 8, 239-250.
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~C ~C~~~

~1 21 ~997 Dellaporta S.L.; J.; Wood, J. and Hicks, B. (1983) Plant Molecular Biology Reports 1, 19-21.
Ellis, J.R., Shirsat, A.H., Hepher, A., Yarwood, J.N., Gate-house, J.A., Croy, R.R.D. and Boulter, D. (1988) Plant Molec-ular Biology 10, 203-214.
Elkin, R.G., and Griffith, J.E. (1985a) J. Assoc. Off. Anal.
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Herman, E.M., Shannon, L.M. and Chrispeels, M.J. (1986) In Molecular Biology of Seed Storage Proteins and Lectins, L.M.
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Higgins, T.J.V., Llewellyn, D., Newbigin, E. and Sr~ncer, D.
(1986) EMBO workshop "Plant Storage Protein Genes~ am and abstract page 19, Eds. J. Brown and G. Feix, University of Freiburg, 1986.
Horsch, R.B., Fry, J.E., Hoffmann, N.L., Eichholtz, D., Rogers, S.G. and Fraley, R.T. (1985) Science 227, 1229-1231.
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Hull and Howell (1987) : Virology, 86, 482-493 Jagodinski, L., Sargent, T., Yang, M., Glackin, C., Bonner, J. (1987) Proc. Natl. Acad. Sci. USA. 78, 3521-3525.

Jones J.D.G.; Dunsmuir, P. and Bedbrook, J. (1985) EMBO J. 4 (10), 2411-2418.

oGC ~C~S~ c~

Joseph, H. and Marsden, J. (1986) "HPLC of Small Molecules -A practical approach" in: IRL Press Oxford - WA~hin~ton D.C.
"Amino Acids and Small Peptides~ Ed.: Kim , C.K., 13-27.
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(1987). J. Biol. Chem. 26~2 (25), 12196-12201.
Klimaszewska, K. and Keller, W.A. (1985) Plant Cell Tissue Organ Culture, 4, 183-197.
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87(4), 859-866.
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~rr ~r~ C)~\~C

M ~ 1 21 i ~0~?~7 - 2 ~ ~ ~ fi 6 ~

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OGC ~

2 ~ 6 ~
M A 1 2 1 ~957 2S Albumin As ~o Of Total Seed Protein Family, spccies %
(common name) Compositae Helianthus artnuus (sunflower) 62 Cruciferae Brassica spp.
(mustard) 62 Llnaceae Linum ~sit~tissimu~n (linseed) 42 Legumlnosae Lupinus polyphyllus (lupin) 38 Arachis hypogaea (peanut) 20 Lecythidaceae BcrthollcRa exçelra (brazil nut) 30 Liliaceae Yucca spp.
(yucca) 27 Euphorbiaceae Ricinus communis <~
(castor bean) 44 (~
From Youle and Huang, 1981

Claims (45)

1. A recombinant DNA encoding a modified 2S albumin with increased nutritional value comprising:
a first nucleic acid sequence encoding a precursor of a 2S albumin from a plant, wherein a second nucleic acid sequence, heterologous with respect to said first nucleic acid sequence, is inserted into or replaces in part a region of said first nucleic acid sequence located between the codons coding for the fourth and the fifth cysteine residue of the large subunit of said 2S albumin, and wherein said second nucleic acid sequence encodes a polypeptide containing at least one essential amino acid, to thereby provide a recombinant DNA sequence encoding a precursor of a modified chimeric 2S albumin which is enriched in at least one essential amino acid as compared to a non-modified 2S albumin.

2. The recombinant DNA of Claim 1, wherein said second nucleic acid sequence is inserted into or replaces in part the region of said first nucleic acid sequence located between the third codon downstream of the codon encoding said fourth cysteine residue and the third codon upstream of the codon encoding said fifth cysteine residue.

3. The recombinant DNA of Claim 1, wherein said second nucleic acid sequence is inserted into or replaces in part the region of said first nucleic acid sequence located between the fourth codon downstream of the codon encoding said fourth cysteine residue and the sixth codon upstream of the codon encoding said fifth cysteine residue.

4. The recombinant DNA of Claim 1, wherein said second nucleic acid sequence is inserted into or replaces in part the region of said first nucleic acid sequence located between the sixth codon downstream of the codon encoding said fourth systeine residue and the sixth codon upstream of the codon encoding said fifth cysteine residue.

5. The recombinant DNA of Claim 1, wherein said first nucleic acid encodes a precursor of a 2S albumin from an Arabidopsis species, a Brassica species, Ricinis communis or Bertholletia excelsa.

6. The recombinant DNA of Claim 5, wherein said first nucleic acid encodes a precursor of a 2S albumin from Arabidopsis thaliana or Brassica napus.

7. The recombinant DNA of Claim 6, wherein said second nucleic acid sequence is inserted into or replaces in part the region of said first nucleic acid sequence located between codon 31 and 57 of the large subunit of the 2S
albumin of Arabidopsis thaliana.

8. The recombinant DNA of Claim 6, wherein said 2S albumin is AT2S1.

9. The recombinant DNA of any one of Claims 1 to 8, wherein said essential amino acid is selected from the group consisting of a lysine, a methionine, a tryptophane, a threonine, a phenylalanine, a leucine, a valine, anarginine and an isoleucine.

10. The recombinant DNA of Claim 9, wherein said second nucleic acid sequence encodes a plurality of said essential amino acids.

11. The recombinant DNA of Claim 10, wherein said second nucleic acid sequence encocles a polypeptide with the sequence GIMMMRMI
or with the sequence GIMMMQPRGDMMMIMMMQPRGDMMMI.

12. The recombinant DNA of any one of Claims 1 to 8, which further comprises an operably linked DNA sequence comprising a plant expressible promoter region.

13. The recombinant DNA of Claim 9, which further comprises an operably linked DNA sequence comprising a plant expressible promoter region.

14. The recombinant DNA of Claim 10, which further comprises an operably linked DNA sequence comprising a plant expressible promoter region.

15. The recombinant DNA of Claim 11, which further comprises an operably linked DNA sequence comprising a plant expressible promoter region.

16. The recombinant DNA of Claim 12, wherein said promoter region is heterologous with respect to said first nucleic acid sequence.

17. The recombinant DNA of Claim 12, wherein said promoter region is naturally associated to said first nucleic acid sequence.

18. The recombinant DNA of Claim 12, wherein said promoter region is a seed-specific promoter region.

19. The recombinant DNA of Claim 13, wherein said promoter region is a seed-specific promoter region.

20. The recombinant DNA of Claim 14, wherein said promoter region is a seed-specific promoter region.

21. The recombinant DNA of Claim 15, wherein said promoter region is a seed-specific promoter region.

22. The recombinant DNA of Claim 18, wherein said promoter region comprises a sequence from figure 4 from nucleotide position -431 to nucleotide position -1.

23. The recombinant DNA of Claim 19, wherein said promoter region comprises a sequence from figure 4 from nucleotide position -431 to nucleotide position -1.

24. The recombinant DNA of Claim 20 wherein said promoter region comprises a sequence from figure 4 from nucleotide position -431 to nucleotide position -1.

25. The recombinant DNA of Claim 21 wherein said promoter region comprises a sequence from figure 4 from nucleotide position -431 to nucleotide position -1.

26. The chimeric 2S albumin encoded by the recombinant DNA of any one of Claims 1 to 8.

27. The chimeric 2S albumin encoded by the recombinant DNA of Claim 9.

28. The chimeric 2S albumin encoded by the recombinant DNA of Claim 10.

29. The chimeric 2S albumin encoded by the recombinant DNA of Claim 11.

30. A plant cell, the genome of said plant cell comprising the recombinant DNA of Claim 12.

31. A plant cell, the genome of said plant cell comprising the recombinant DNA of Claim 13.

32. A plant cell, the genome of said plant cell comprising the recombinant DNA of Claim 14.

33. A plant cell, the genome of said plant cell comprising the recombinant DNA of Claim 15.

34. A process for producing a plant with increased nutritional value which comprises transforming the genome of a plant with the recombinant DNA
of Claim 12.

35. A process for producing a plant with increased nutritional value which comprises transforming the genome of a plant with the recombinant DNA
of Claim 13.

36. A process for producing a plant with increased nutritional value which comprises transforming the genome of a plant with the recombinant DNA
of Claim 14.

37. A process for producing a plant with increased nubitional value which comprises transforming the genome of a plant with the recombinant DNA
of Claim 15.

38. The process of Claim 34, wherein said plant belongs to the genenus Arabidopsis or Brassica.

39. The process of Claim 35, wherein said plant belongs to the generus Arabidopsis or Brassica.

40. The process of Claim 36, wherein said plant belongs to the generus Arabidopsis or Brassica.

41. The prooess of Claim 37, wherein said plant belongs to the generus Arabidopsis or Brassica.

42. The process of Claim 38, wherein said plant is Brassica napus.

43. The process of Claim 39, wherein said plant is Brassica napus.

44. The process of Claim 40, wherein said plant is Brassica napus.

45. The process of Claim 41, wherein said plant is Brassica napus.