WO2002086077A2 - Co-expression of zein proteins - Google Patents

Abstract

Methods and constructs for transformed plants and plant tissues that are capable of expressing high levels of stable proteins localized as protein bodies within the plant cell. Transformed plants co-expressing high levels of both the 15 kD and 10 kD zein proteins are disclosed which accumulate to high levels as protein bodies in the vegetative tissue of the plant. Transformed plants co-expressing the 15 kD and 10 kD zein proteins are useful for providing forage crops containing increased levels of sulfur containing amino acids, such as methionine, in the diet of animals that normally feed on such crops. In one embodiment, a stable protein body is expressed in a plant or plant tissue as a fusion protein comprising both the 15 kD and 10 kD zein proteins operably linked by a polypeptide or peptide linker. The protein bodies provided in the present invention are resistant to rumin digestion or environmental degradation.

Description

CO-EXPRESSION OF ZEIN PROTEINS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing of U S Provisional Patent Application Serial

No 60/284,732, entitled "Co-Expression of 15 kD and 10 kD Zein Proteins", filed on April 18, 2001 , and the specification thereof is incorporated herein by reference

This application is a continuation-in-part application of U S Patent Application Serial No 09/479,724, entitled "Co-Expression of Proteins", to Suman Bagga, Champa Sengupta-Gopalan and John D Kemp, filed on January 7, 2000, which is a division of application Serial No 09/224,655, filed December 31 , 1998, now abandoned, which is a continuation-in-part application of application Serial No 08/866,879, now United States Patent No 5,990,384, issued November 23, 1999, and provisional application Serial No 60/020,424, filed May 31 , 1996, now abandoned, and the specification thereof of each is incorporated herein by reference

GOVERNMENT RIGHTS The U S Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Grant No 99-34331-7524 and Grant No 99-34250-7420, both awarded by the United States Department of Agriculture

BACKGROUND OF THE INVENTION Field of the Invention (Technical Field) The present invention relates to methods and constructs for co-expression of zein proteins in plants, including forage plants, including co-expression of a 15 kD and 10 kD zein protein by means of a gene construct coding for such proteins and for a linker peptide sequence joining such proteins Backqround Art

Note that the following discussion refers to a number of publications by author(s) and year of publication, and that due to recent publication dates certain publications are not to be considered as prior art vis-a-vis the present invention Discussion of such publications herein is given for more complete background and is not to be construed as an admission that such publications are prior art for patentability determination purposes

Alfalfa (Medicago sativa L ) is considered to be the most important cultivated forage crop in the world It is widely grown, has a superb balance of vitamins and minerals, is high yielding, is an excellent source of biological nitrogen fixation, and it serves as an attractive nectar source for honeybees Alfalfa has been bred for years for both forage quality and plant performance Although alfalfa and other leguminous forage crops are high in protein, these plants are deficient in the sulfur ammo acids (S-amino acids), methionine and cysteine It has been shown that wool growth in sheep is limited by the availability of S-amino acids Similarly, milk production by dairy animals is affected by the deficiency of S-amino acids in plants Efforts to use conventional plant breeding and cell selection techniques to increase the S-amino acid content of alfalfa have met with little or no success

A genetic engineering approach to improve the ammo acid balance of alfalfa and other forage crops would be to introduce into these plants genes encoding proteins high in methionine driven by a strong constitutive promoter or a leaf promoter In order to significantly alter the ammo acid balance of legume forage, the foreign proteins should contain about 15 to 25% of S-amino acids and constitute 5 to 10% of the total leaf protein To achieve these levels of protein accumulation, one has to ensure not only maximum levels of transcription and translation of the gene but also the stability of the protein In regard to forage crops for ruminant animals, the digestibility of S-amino acid containing proteins by the rumen bacteria and the stomach enzymes is also an extremely critical issue in regard to providing a suitable forage crop for ruminant animals, but is often overlooked Thus, the S-amino acid rich protein should be relatively resistant to degradation in the rumen (first stomach) of the ruminant animals and should be assimilated in the lower gastrointestinal tract Most of the concerted efforts in regard to nutritional improvement in plants have focused on seed proteins Since corn and other cereal crops are not easily transformable, most work directed to seed protein modification has involved testing stability of modified prolamine proteins in transgenic tobacco and Xenopus oocytes The synthesis of lysine containing α zeins was also analyzed in transgenic tobacco and petunia seeds Both the normal and modified proteins were found to have a very short half-life

Efforts to improve the S-amino acid content of legume seed proteins have included introducing a 45 bp o gonucleotide containing six methionine codons into the third exon of a β- phaseohn gene Transformants containing this modified gene showed that the high methionine phaseo n was synthesized at the same level as the normal protein, but was very unstable and was rapidly turned over Introduction of the extra ammo acids in the β-phaseolm protein probably caused a distortion in its secondary structure making it more susceptible to proteolytic degradation DeClercq et al (DeClercq, A , M Vandewlele, R De Rycke, J Van Damme, M Van aMontagu, E Krebbers, J Vandekerckjove (1990) "Expression and processing of an Arabidopsis 2S albumin in transgenic tobacco" Plant Physiol 94 970-979) replaced a 23 ammo acid coding segment between the sixth and seventh cysteme residues of Arabidopsis 2S albumin, with three different high methionine coding fragments These modified Arabidopsis 2S genes were transformed into A thaliana, B napus and tobacco There was some accumulation of the protein in the seeds but not as much as predicted The gene of the 2S albumin of Brazil nut, which contains up to 19% methionine, and driven by the β-phaseolm gene promoter, has been introduced into tobacco, rape and soybean Saalbach et al (Saalbach, I , et al (1994) "A chimeπc gene encoding the methionine-nch 2S albumin of the Brazil nut {Bertholletia excelsa H B K ) is stably expressed and inherited in transgenic gram legumes" Mol Gen Genet 242 226-236) synthesized the 2S albumin gene and engineered it behind the CaMV 35S promoter The gene, when introduced into tobacco and some grain legumes, showed the highest level of expression in the plant leaves with the protein localized in vacuoles However, Brazil nut albumin protein is extremely allergenic and may not be acceptable for consumption U S Patent No 5,939,599, to Chui et al , issued August 17, 1999, discloses a nucleic acid fragment for overexpression of a high methionine seed storage protein in plants, which nucleic acid fragment is derived from and related to the 10 kD δ zein

Another approach to increase the pools of particular ammo acids in plants has been to introduce bacterial genes encoding for key regulatory enzymes in ammo acid biosynthetic pathways in plants A bacterial gene encoding for aspartate kinase which is desensitized to feedback inhibition by lysine and threonme was fused to the β-phaseohn gene promoter and introduced into tobacco The seeds of the transgenic tobacco showed increased levels of free threonme and methionine

Very little effort has been made with regards to improving forage crop protein quality

Schoeder et al (Schroeder, H E , et al (1991 ) "Expression of a chicken ovalbumin gene in three lucerne cultivars" Aust J Plant Physiol 18 495-505) introduced the chicken ovalbumin gene (cDNA), driven by a CaMV 35S promoter, into alfalfa The transgenic alfalfa plants, however, showed very low level accumulation of the protein in the leaves (0 005%) The basis for such a low abundance of this protein in the transgenic alfalfa leaves was not determined

Some efforts to obtain alfalfa mutants that have larger free methionine levels have also been attempted at the University of Wisconsin Cell lines with resistance to growth inhibition by an ammo acid analogs reportedly produce higher than normal amounts of the corresponding natural am o acid Hence, growth on specific ammo acid analogs has been used as a selection tool to select for plants accumulating high levels of a particular ammo acid Ammo acid over-production is usually due to relaxed feedback control of an enzyme involved in its production In an attempt to improve the methionine content of alfalfa, mutagenized suspension culture cells of alfalfa were selected for resistance to growth inhibition by a methionine analog A few cell lines containing high methionine pools were obtained, however, regeneration of these cell lines did not produce plants with high methionine content

Zems are a group of alcohol soluble proteins that are synthesized during endosperm development in corn and constitute 50% of the total protein in mature seeds The zems can be divided into four groups, the α, β, y and δ, based on their solubility (Larkms, B A , C R Lending, J C Wallace, G Galili, E E Kawata, K B Geetha, A L Kirz, D M Martin, and C E Bracker (1989) "Zein gene expression during maize endosperm development" In Goldbert R B (Ed) The Molecular Basis Of Plant Development, Alan R Liss, NY pp 109-120) The zems can also be separated by size into groups The α zems, which are the most abundant class, are made up of the 22 kD and 19 kD ze s, the central region of these proteins consists of repetitive peptides of about 20 ammo acid residues The β zems comprise the 15 kD zein which contains less prolme and glutamme than the α zems The y zems include the 27 kD and 16 kD class and are very rich in prolme (25%) The δ zems are a relatively minor class consisting of the 10 kD zein All the zein classes are structurally unique The repeat regions in the α and y zems probably have a major role in the packing of protein bodies Zems, in general, contain extremely low levels of the essential ammo acids lysme, tryptophan and to a lesser extent methionine The 15 kD and 10 kD zems, however, are distinguished by their extremely high methionine content (10% and 22 5%, respectively)

The zems are synthesized on the endoplasmic reticulum (ER) and they aggregate into protein bodies directly in the ER Based on the analysis of the zein composition of developing protein bodies in corn endosperm, Lending and Larkms (Lending, C R and B A Lark s (1989) "Changes in the zein composition of protein bodies during maize endosperm development" Plant Cell 1 1011-1023), have proposed a descriptive model for the pattern of zein deposition during protein body formation in corn endosperm the β and y zems are the first to start accumulating within the ER Subsequently, α zems begin to accumulate as locules within the β and y zems With time, the α zein locules fuse and form a central core while the β and y ze s form a continuous layer around the periphery of the protein body In a separate study, Esen and Stetter (Esen, A and D A Stetter (1992) "Immunochemical location of γ-zeιn in the protein bodies of maize endosperm" Am J Bot 79 243-248), demonstrated that the δ zein occurs throughout the core region of the protein body

Mutations in maize affect the expression of the different zein genes Changes in zein gene expression in turn have direct impact on the ammo acid composition of the seeds Seeds of plants homozygous for the recessive mutation opaque-2, have increased levels of lysme compared to the wild-type seeds The increase in lysme is due to the reduced expression of the 22 kD α zems The inbred line BSSS-53 has 30% higher level of seed methionine compared to other inbred lines This increase in methionine content is because of a two-fold increase in the level of the 10 kD zein

Proteins that accumulate in the endoplasmic reticulum are known to have the ammo acid sequence Lys(Hιs)-Asp-Glu-Leu (K(H)DEL) (SEQ ID NO 1 ) near their carboxy terminal end which prevents them from exiting into the Golgi The zems and other prolammes, however, lack this sequence A cognate of the 70-kD heat shock protein, BiP, which functions as a molecular chaperone, has been shown to be involved in the formation of prolamme protein body formation in rice endosperm The determination of involvement of BiP in the formation of zein protein bodies is based on the fact that BiP accumulates to high levels in the ER and on the abnormal protein bodies of some of the zein regulatory mutants of corn Overall, however, the mechanisms of zein targeting and assembly in protein bodies are poorly understood and it is not known whether inter- and intramolecular interactions play a key role in protein body formation

U S Patent No 5,990,384 discloses and claims a method to modify plants such that the resulting transgenic plant expresses both a 15 kD zein, such as a β zein, and a 10 kD zein, such as a δ zein In one embodiment, this is accomplished by introducing 15 kD or 10 kD zein coding sequences under the control of a promoter, such that the resulting transgenic plant expressed either the 15 kD or the 10 kD zein, and thereafter breeding sexual crosses The resulting seeds produce seedlings expressing both genes Genetic constructs can further be made expressing both the 15 kD and the 10 kD zein (S Bagga et al (1997) "Co-expression of the maize δ-zein and β-zein genes results in stable accumulation of δ-zem in endoplasmic reticulum-deπved protein bodies formed by β-zein" Plant Cell 9 1683-1696) U S Patent Application Serial No 09/479,724 discloses a specific chimeπc gene, consisting of a 10 kD zein gene fused in frame to the front of an oryzacystatm I protease inhibitor gene, resulting in a construct producing proteins that both provide S-amino acids and control plant pests The chimeπc gene includes both the coding and signal peptide regions of the 10 kD zein The 10 kD zein protein, which contains an N-termmal signal peptide that directs the protein into the ER, is folded into a tertiary conformation depending, in part, on the signal peptide Modification of the signal peptide can result in varying accumulation patterns, such that a morphologically distinct protein body results (J Randall et al (2000) "A modified 10 kD zein protein produces two morphologically distinct protein bodies in transgenic tobacco" Plant Science 150 21-28)

As can be understood from the above, there remains a need in the art for plants and forage crops that contain stable protein bodies that are high in S-amino acid content The subject invention provides a novel and advantageous means for improving the forage quality of plants

SUMMARY OF THE INVENTION (DISCLOSURE OF THE INVENTION)

The invention provides an isolated and purified nucleic acid fragment, which fragment includes a nucleotide sequence encoding a 15 kD zein and a nucleotide sequence encoding a 10 kD zein, joined, directly or through one or more additional sequences, by a nucleotide sequence encoding a polypeptide linker sequence In one embodiment, the polypeptide linker sequence is a signal peptide for one of the encoded ze s, and is more specifically a signal peptide for a 10 kD zein In the nucleic acid fragment, the codon coding the polypeptide linker sequence is preferably from about ten to about thirty ammo acid residues, and is more preferably about twenty ammo acid residues The nucleic acid fragment may optionally include the nucleotide sequence of either SEQ ID NO 4 or SEQ ID NO 6 In one embodiment, the nucleic acid fragment is operably linked to a promoter, a plant signal sequence, or a regulatory sequence directing expression in one or more organs of a plant, or any combination of the foregoing

In another embodiment, the invention provides a transgenic plant transformed with a gene sequence encoding a 15 kD zein and a nucleotide sequence encoding a 10 kD zein, joined, directly or through one or more additional sequences, by a nucleotide sequence encoding a polypeptide linker sequence, and operably linked to a regulatory sequence directing expression in one or more organs of a plant The invention further provides a polypeptide product including a 15 kD zein linked to a 10 kD zein by a polypeptide linker, wherein the polypeptide linker includes from about ten to about thirty ammo acid residues The polypeptide product may result from the expression in a prokaryotic or eukaryotic host cell of a nucleic acid fragment as described above

In yet another embodiment, the invention provides a method for expression of stable protein bodies in a plant, the method including transforming a plant or plant tissue with a polynucleotide molecule that encodes a storage protein comprising a first protein and a second protein joined by a polypeptide linker, wherein the storage protein is expressed and accumulated as a protein body in a vegetative tissue of the plant or plant tissue In this method, the first protein and second protein may ^' be zein proteins, and in any event, the first protein and second protein may be different If the first protein and second protein are zein proteins, they may be a 10 kD zein protein and a 15 kD zein protein The storage protein made by the method may, in a preferred embodiment, be rumm stable

The invention further provides a composition that includes a rumm stable protein body, wherein said protein body comprises a first protein and a second protein joined by a polypeptide linker, and wherein the protein body is expressed and accumulated in a plant In this composition, the first protein and second protein can be zein proteins

A primary object of the present invention is to provide a fusion protein, composed of two zein proteins joined by a linker, to increase availability of S-amino acids in plants

Another object of the present invention is to provide a gene fusion construct, which construct includes one or more promoters, and further includes sequences coding for two zein proteins and a linker

Another object of the invention is to provide transgenic plants wherein leaves and/or seeds contain stable protein bodies, the protein bodies including a protein consisting of two zein protein sequences joined by a linker polypeptide Yet another object of the invention is to provide stably transformed plants, such that seeds derived from such plants contain nucleic acid sequences coding for two zein proteins and a linker Preferred are plants with utility as forage plants, such as alfalfa, but other plants are included, such as corn, soybeans, rapeseed, tobacco and rice

A primary advantage of the present invention is that it provides a single linked protein, consisting of two zein proteins joined by a polypeptide linker, which protein can be expressed in commercially useful quantities in plants

Another advantage of the present invention is that, by means of the polypeptide linker, the two linked zein proteins may each assume a desired tertiary structure

Another advantage of the present invention is that the expression of S-amino acid proteins and protein bodies is enhanced by means of two zein proteins joined by a polypeptide linker, as compared to two zein proteins directly joined without a polypeptide linker

Other objects, advantages and novel features, and further scope of applicability of the present invention will be set forth in part in the detailed description to follow, taken in conjunction with the accompanying drawings and sequence listings, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated into and form a part of the specification, illustrate one or more embodiments of the present invention and, together with the description, serve to explain the principles of the invention The drawings are only for the purpose of illustrating one or more preferred embodiments of the invention and are not to be construed as limiting the invention In the drawings FIG 1 shows a diagrammatic representation of a fusion gene construct of a 15 kD β zein and a 10 kD δ zein without a linker

FIG 2 shows a diagrammatic representation of a fusion gene construct of a 15 kD β zein and a 10 kD δ zein with a linker, the linker depicted as the signal peptide (SP) gene sequence for the 10 kD δ zein

FIG 3 shows the western analysis of independently transformed tobacco plants accumulating the β/δ zein fusion proteins Fifty micrograms of ethanol soluble protein was separated on a 16% SDS-PAGE gel, transferred to nitrocellulose, and immunodetected with β and δ zein antiserum The top blot was immunodetected with a δ zein antibody and the bottom blot with a β zein antibody Lanes 1 , 2, 3, and 4 are protein from transgenic plants containing the β/δ zein fusion construct without a linker Lanes A, B, and C are protein from transgenic plants containing the β/δ zein fusion construct with a linker of the construct of FIG 2, including the linker sequence of SEQ ID NO 5 Lane NT is the non-transformed negative control The positive control on the top blot is a high accumulating altered 10 kD zein transgenic tobacco plant, and the positive control on the bottom blot is a high accumulating 15 kD zein transgenic tobacco plant As depicted, protein levels in lanes A and C are higher than lanes 1 , 2, 3, and 4 β/δ zein fusion protein accumulates to higher levels in plants A and C than in plants 1 , 2, 3, and 4

FIG 4 shows an electron micrograph of leaves from independently transformed tobacco plants accumulating the β/δ zein fusion proteins with a linker of the construct of FIG 2, including the linker sequence of SEQ ID NO 5, with localization using anti-β-zein polyclonal rabbit antisera bound to gold Leaves from transformed plants were sectioned and fixed as described by Bagga et al , 1995 The grids were observed using a Hitachi H700 transmission electron microscope In the micrograph, "RB" depicts πbosomes, "PB" depicts protein body, "CW" depicts cell wall and "MT" depicts mitochondria, and the arrows indicate the gold-conjugated antibody FIG 5 shows an electron micrograph as in FIG 4, but with localization using anti-δ-zem polyclonal rabbit antisera In the micrograph, "PB" depicts protein body, "RB" depicts nbosomes and "CP" depicts chloroplasts, and the arrows indicate the gold-conjugated antibody

FIG 6 shows the fusion gene construct and coded proteins for the fusion portion of a 15 kD β zein and a 10 kD δ zein without a linker The 5' end through position 6 is the β zein, the 3' end from position 13 to 30 is the δ zein, and the position 7 to 12 are two ammo acids, Arg and Ser, introduced with the restriction site

FIG 7 shows the fusion gene construct and coded proteins for the fusion portion of a 15 kD β zein and a 10 kD δ zein with a linker, wherein the linker includes the signal peptide gene sequence for the 10 kD δ zein The 5' end through position 6 includes a portion of the β zein, the 3' end from position 73 to 87 includes a portion of the δ zein, position 7 to 12 code for two am o acids, Arg and Ser, introduced with the restriction site, and position 13 to 72 denotes the linker, here the signal peptide sequence for the 10 kD δ zein

FIG 8 shows the fusion gene construct and coded proteins for the fusion portion of a 15 kD β zein and a 10 kD δ zein with a linker The 5' end through position 6 includes a portion of the β zein, the 3' end from position 73 to 87 includes a portion of the δ zein, position 7 to 12 code for two ammo acids, Arg and Ser, introduced with the restriction site, and position 13 to 72 denotes the linker

BRIEF DESCRIPTION OF THE SEQUENCES The accompanying sequence listing, which is incorporated into and forms a part of the specification, illustrate one or more embodiments of the present invention and, together with the description, serve to explain the principles of the invention The sequences are only for the purpose of illustrating one or more preferred embodiments of the invention and are not to be construed as limiting the invention In the sequence listing SEQ ID NO 1 represents a plant protein sequence found in proteins that accumulate in the endoplasmic reticulum, which sequence is near the carboxy terminal end of the protein and serves to prevent the protein from exiting into the Golgi

SEQ ID NO 2 represents the fused gene construct of FIGS 1 and 6

SEQ ID NO 3 represents the ammo acid sequence coded by SEQ ID NO 2

SEQ ID NO 4 represents the fused gene construct of FIGS 2 and 7

SEQ ID NO 5 represents the ammo acid sequence coded by SEQ ID NO 4

SEQ ID NO 6 represents the fused gene construct of FIG 8

SEQ ID NO 7 represents the ammo acid sequence coded by SEQ ID NO 6

DESCRIPTION OF THE PREFERRED EMBODIMENTS (BEST MODES FOR CARRYING OUT THE INVENTION) The subject invention concerns plants and plant tissues that are capable of expressing high levels of stable storage proteins that are localized as protein bodies within the plant cell Plants contemplated within the scope of the invention include forage crop plants, including, for example, alfalfa, clover, corn silage, sorghum and other leguminous crops, transformed to express the proteins of the invention Also contemplated within the scope of the present invention are plants for human consumption, which have been transformed to express proteins that enhance the protein quality of the plant for improved nutrition Specifically exemplified are plants expressing proteins containing high levels of S-amino acids, such as methionine and cysteine In a preferred embodiment, a zein protein is expressed in the plant or plant tissue More preferably, the zein protein expressed is the 15 kD and 10 kD zein proteins co-expressed in the plant or plant tissue, preferably joined by a polypeptide linker The zein proteins expressed in plants are preferably resistant to rum degradation and, therefore, are useful for providing nutritionally important ammo acids that can be digested in the stomach and absorbed by the ruminant animal because of the protein's capacity to "by-pass" the rumm

Also contemplated by the subject invention are plants or plant tissue comprising rumm stable protein bodies which contain other prote aceous material, for example, an antigenic determinant capable of eliciting an immune response, a protemaceous drug, pesticide or antimicrobial peptide Heterologous and endogenous proteins and synthetic peptides having essential ammo acids can be expressed in plants transformed with the storage proteins of the subject invention which can act as a "carrier protein," whereby the proteins coalesce and accumulate in the cell as a protein body In an alternative embodiment, a rumm stable protein body is expressed in a plant or plant tissue as a fusion protein comprising a zein protein and a heterologous protein or peptide The fusion protein can be designed to yield the heterologous protein portion by cleavage with a selected enzyme or under certain physiological conditions Preferably, both the 15 kD and 10 kD zein proteins are co- expressed in the plant or plant tissue comprising the fusion protein, joined by a linker, such as a polypeptide linker

The subject invention also pertains to a rumm stable protein body Rumm stable protein bodies of the invention are not subject to digestion by rumm bacteria in the rumm of an animal but can be digested proteolytic enzymes of an animal's stomach A rumm stable protein body of the present invention can be prepared which contains heterologous protemaceous material in addition to the rumm stable protein, for example, an antigenic determinant capable of eliciting an immune response, a protemaceous drug, pesticide or antimicrobial peptide Rumm stable protein bodies can be isolated from plants that have been transformed with polynucleotide molecules encoding the desired rumm stable proteins Plant cells expressing the polynucleotide molecules encoding the desired rumm stable proteins can be readily selected and regenerated into plants or plant tissue using standard techniques known in the art ln one embodiment of the present invention, a first storage protein gene is co-expressed in a cell with a second storage protein gene whereby the first gene and second gene are joined by a linker, such that the first protein and second protein are joined by a polypeptide linker Thus in one embodiment a gene fusion construct is provided, wherein the β zein coding region is fused to the δ zein coding region with a linker coding region between the genes The gene fusion construct can be introduced into a plant to be transformed behind a suitable promoter, such as the 35S constitutive promoter The resulting fusion protein construct with a linker between the two zein proteins accumulates to higher levels than a protein construct without a linker Further, plants with the fusion protein containing the linker produce protein bodies, which protein bodies are different morphologically than the rosette, spherical or aggregate-shaped protein bodies previously noted with zein proteins

The linker in the gene fusion construct is selected from any suitable sequence coding for a polypeptide that may be employed as a linker In one embodiment, the linker is a signal peptide gene sequence for a zein protein, such as the signal peptide for the δ zein In this embodiment, the expressed signal peptide sequence does not necessarily serve as a functional signal peptide, but rather serves to link the β zein and the δ zein The linker may alternatively be any sequence coding a utilizable fusion polypeptide Thus, for example, the linker polypeptide, and gene coding therefore, disclosed by Prescott et al may be employed (Prescott, M et al (1999) "The length of polypeptide linker affects the stability of green fluorescent protein fusion proteins" Anal Biochem 273 305-307) It is hypothesized that the resulting linker polypeptide should be of such length as to permit the two fusion zein proteins to each assume a desired and typical tertiary structure It is further hypothesized that two fusion zein proteins linked without a linker sequence do not assume a desired and typical tertiary structure, because of steric and related considerations, thereby adversely affecting the stability of the protein and the ability of the protein to form protein bodies In general, the linker polypeptide may be of any desired length, such as from about ten to about thirty am o acids, and preferably about twenty ammo acids Regulatory sequences employed with the protein genes (promoters, initiation sequences, termination sequences, polyadenylation sequences, enhancers, etc ) are readily chosen by one of ordinary skill in the art based on a variety of factors, such as, for example, i) the specific protein genes employed, n) the target cell to be transformed, in) the plant tissue where expression/accumulation is desired, iv) the particular plant (monocot, dicot, etc) species to be transformed, etc For example, when a plant cell is the target cell then a constitutive promoter may be chosen (e g , CaMV 35S, ubiquitm, etc ) or a tissue specific promoter may be employed that will express at high levels in specific tissues (seeds, green tissues, etc ) In general, promoters that can be employed with ze s include, in addition to the foregoing, the CvMV promoter, a constitutive promoter from cassava, and the phaseolm promoter

In a preferred embodiment of the present invention, alfalfa, tobacco or other plant cells are transformed with a fusion gene construct composed of a 15 kD zein protein gene and a 10 kD zein protein gene, joined by a sequence coding a linker polypeptide, wherein both zein genes are driven by a constitutive promoter Fertile, transgenic plants containing the fusion gene construct are regenerated Progeny plants are grown and the 10 kD zein protein is accumulated in green tissue at levels significantly more than the accumulation level of the 10 kD protein when expressed alone, the 15 KD protein when expressed alone, or a construct of the 10 kD and 15 kD protein not joined by a polypeptide linker

Additionally, the present invention encompasses novel protein bodies formed as a result of expressing a fusion protein gene construct in green plant tissues In one embodiment, the novel protein body includes 10 kD zein protein segment joined, by means of a polypeptide linker, to a 15 kD zein protein segment The protein body is typically located in leaf tissue In a preferred embodiment, the novel protein body is located in leaf tissue and comprises a 15 kD zein protein segment and a 10 kD zein protein segment linked by means of a polypeptide linker

The subject invention also concerns a method for increasing the forage quality of a plant comprising transforming a plant or plant tissue with a polynucleotide molecule that encodes a storage protein of the present invention Methods for transforming plants and selecting for expression of the transformed genotype are known in the art In a preferred embodiment of the method, the polynucleotide encodes a zein protein which is expressed in the plant or plant tissue More preferably, the zein protein expressed is the 15 kD and the 10 kD zein protein Most preferably, the zein protein expressed is the 15 kD and 10 kD zein proteins joined by a polypeptide linker, and co- expressed in the transformed plant or plant tissue Transgenic plants can be readily prepared from the transformed plant or plant tissue using standard techniques known in the art

It is known that β and δ zems produce ER derived protein bodies in the leaves and seeds of transgenic tobacco plants when under the control of the 35S constitutive promoter Both proteins are stable and accumulate to high levels in the leaves of transgenic tobacco plants ER-deπved protein bodies have been observed in the leaves of these transgenic tobacco plants containing the δ (10 kD) and β (15 kD) genes Morphology differences of the ER derived protein bodies in leaves have been observed between the δ and β zein transgenic plants The β zein protein immunolocalizes in rosette- shaped ER protein bodies and the δ zein protein immunolocalized in spherical-shaped ER protein bodies When plants containing the β zein protein are crossed with plants containing the δ zein protein, co-localization of both proteins is found in the β zein rosette bodies (Randall et al , 2000) The protein bodies produced by a fusion protein consisting of β and δ zems joined by a polypeptide linker appear to have a slightly different morphology than the rosette or spherical bodies previously observed

The zein proteins of the present invention include not only those proteins having the same ammo acid sequence as found in nature, including alle c variants, but also includes those variant zein proteins having conservative ammo acid substitutions, additions and deletions in the protein sequence, as long as the variant protein retains substantially the same relevant biological activity as the native zein protein The skilled artisan, having the benefit of the teachings disclosed herein, can readily determine whether a variant protein retains the substantially the same biological activity as the non-modified protein Standard procedures can be employed for recombinant DNA manipulations Plasmid pMZEHOk containing the 10 kD zein cDNA isolated from a corn endosperm cDNA library (Kiπhara, J A , et al (1988) "Differential expression of a gene for a methionine-nch storage protein in maize" Mol Gen Genet 211 477-484), was a gift from Dr J Messing A 470 bp EcoR1/Xba1 fragment containing the entire coding region was removed from pUC 119 and cloned into the EcoRI and Xba1 sites of pSP73 The stop codon for the 10 kD zein is contained within the Xba1 site The 10 kD zein gene was then recovered as a Bglll/Xhol fragment and inserted into the Bglll and Xhol sites in the poly nker of pMON316 (Rogers et al , 1987) The translation terminator following the stop codon of the 10kD zein is the NOS terminator The resulting plasmid was called pM10Z Plasmid pMEZ, containing the 15 kD zein cDNA, is as described by Bagga et al (Bagga, S , Adams Hanke, J D Kemp, and C Sengupta-Gopalan (1995) "Accumulation of 15 kD zein in novel protein bodies in transgenic tobacco" Plant Physiol 107 13-23)

The invention is further illustrated by the following non-limiting examples

Example 1

The stop and 3' UTR were removed from the β (15 kD) zein gene and a Bgl II site was added to the 3' end for cloning purposes The δ (10 kD) mature gene consists of the site from the mature coding sequence to the natural stop with the addition of a Bgl II at the 5' end of the gene for cloning purposes The construct is graphically illustrated at FIG 1 , and the sequence is shown at FIG 6, with the sequence of the gene at SEQ ID NO 2 and the resulting ammo acid sequence at SEQ ID NO 3

Example 2

The full length δ (10 kD) zein gene including its signal peptide was placed behind the β (15 kD) zein with no stop at the Bgl II site The signal peptide is used as a 20 ammo acid linker between the two zein proteins to maintain their tertiary configuration Constructs are placed in pGG prior to plant transformation The construct is graphically illustrated at FIG 2, and the sequence is shown at FIG 7, with the sequence of the gene at SEQ ID NO 4 and the resulting ammo acid sequence at SEQ ID NO 5 Example 3

Transfer of the constructs of Examples 1 and 2 into Agrobacteπum tumefaciens (strain pTιT37ASE) was accomplished through tπ-parental matmgs Nicotiana tabacum plants were transformed using the leaf disc transformation system Transfomants were selected on MS media containing 100 μg/mL Kanamycm and the presence of the genes in transgenic tobacco was confirmed by PCR or Nopaline analysis

Example 4 Protein was isolated from leaf tissue of transformed plants of Example 3 as described by

Bagga et al (1995) Zein proteins were detected after separating 50 μg of ethanol soluble protein on a 16% SDS-PAGE gel and challenging the blot with anti-β-zem polyclonal rabbit antisera or anti-δ- zein polyclonal rabbit antisera

Example 5

Leaves from transformed plants of Example 3, using the construct of Example 2 including a signal peptide linker sequence, were sectioned and fixed Detection was using gold-conjugated anti- β-zein polyclonal rabbit antisera or anti-δ-zem polyclonal rabbit antisera The grids were observed using a Hitachi H700 transmission electron microscope As shown in FIGS 4 and 5, protein bodies specific for both β zein and δ zein were detected

Example 6

A synthetic linker containing a Bgl II site is synthesized to the 5' end of the 10 kD zein mature coding sequence This synthetic linker with the 10 kD mature coding sequence is then placed behind the 15 kD zein, with no stop, at the Bgl II site The resulting construct is then placed in pGG prior to plant transformation, resulting in a sequence as shown in SEQ ID NO 6 The preceding examples can be repeated with similar success by substituting the geneπcally or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples

Although the invention has been described in detail with particular reference to these preferred embodiments, other embodiments can achieve the same results Variations and modifications of the present invention will be obvious to those skilled in the art and it is intended to cover in the appended claims all such modifications and equivalents The entire disclosures of all references, applications, patents, and publications cited above are hereby incorporated by reference

Claims

What is claimed is

1 An isolated and purified nucleic acid fragment comprising a nucleotide sequence encoding a 15 kD zein and a nucleotide sequence encoding a 10 kD zein, joined by a nucleotide sequence encoding a polypeptide linker sequence

2 The nucleic acid fragment of claim 1 , wherein the polypeptide linker sequence is a signal peptide for one of the encoded zems

3 The nucleic acid fragment of claim 2, wherein the polypeptide linker sequence is a signal peptide for a 10 kD zein

4 The nucleic acid fragment of claim 1 , wherein the polypeptide linker sequence comprises codons coding from about ten to about thirty ammo acid residues

5 The nucleic acid fragment of claim 4, wherein the polypeptide linker sequence comprises codons coding about twenty ammo acid residues

6 The nucleic acid fragment of claim 1 , comprising a nucleotide sequence selected from the group consisting of SEQ ID NO 4 and SEQ ID NO 6

7 The nucleic acid fragment of claim 1 operably linked to a promoter

8 The nucleic acid fragment of claim 1 operably linked to a plant signal sequence

9 A gene sequence comprising the nucleic acid fragment of claim 1 operably linked to a regulatory sequence directing expression in one or more organs of a plant 10 A transgenic plant transformed with the gene sequence of claim 9

11 A polypeptide product comprising a 15 kD zein linked to a 10 kD zein by a polypeptide linker, wherein the polypeptide linker comprises from about ten to about thirty ammo acid residues

12 A polypeptide product of the expression in a prokaryotic or eukaryotic host cell of a nucleic acid fragment of claim 1

13 A method for expression of stable protein bodies in a plant, comprising transforming a plant or plant tissue with a polynucleotide molecule that encodes a storage protein comprising a first protein and a second protein joined by a polypeptide linker, wherein the storage protein is expressed and accumulated as a protein body in a vegetative tissue of the plant or plant tissue

14 The method of claim 13 wherein the first protein and second protein are zein proteins

15 The method of claim 13 wherein the first protein and second protein are different

16 The method of claim 14 wherein the zein proteins are a 10 kD zein protein and a 15 kD zein protein

17 The method of claim 13 wherein the storage protein is rumm stable

18 A composition comprising a rumm stable protein body, wherein said protein body comprises a first protein and a second protein joined by a polypeptide linker, and wherein the protein body is expressed and accumulated in a plant

19 The composition of claim 18 wherein the first protein and second protein are zein proteins 20 The composition of claim 18 wherein the first protein and second protein are different

21 The composition of claim 19 wherein the zein proteins are a 10 kD zein protein and a kD zein protein