Patent Application
20070277263
The potato type two inhibitors are a family of serine proteinase inhibitors that are found in many Solanaceous plants. The inhibitors are so named because the first members described were isolated from potato and tomato plants [Bryant, J. et al. (1976) Biochemistry 15:3418-3424; Plunkett, G. et al. (1982) Arch. Biochem. Biophys. 213:463-472]. The inhibitors often consist of two repeated domains each domain of about 6 kDa and with a reactive site to either chymotrypsin or trypsin. These two-domain inhibitors are encoded by genes, termed the Pin2 gene family, which are expressed in tomato fruit and potato tubers, as well as in the leaves of both plants after mechanical wounding or insect damage [Graham, J S, et al. (1985) J. Biol. Chem. 260:6561-6564; Keil, M. et al. (1986) Nuc. Acids Res. 14:5641-5650; Thornberg, R W, et al. (1987) Proc. Natl. Acad. Sci. USA 84:744-748]. Several members of this gene family have been cloned from potato and tomato and most have the same two-domain structure as the original members described [Sanchez-Serrano, J. et al. (1986) Mol. Gen. Genet. 203:15-20; Thornberg supra].
The potato type two inhibitors are referred to simply as “type two” inhibitors herein. Type two inhibitors are structurally related proteins that are encoded by a family of genes knows as Pin2. At least 11 homologous Pin2 genes have been found in both mono- and di-cotyledonous plants. Pin 2 genes can encode either a single 6 kDa proteinase inhibitor (PI) domain, two 6 kDa PI domains like those that are common in potato and tomato or several highly homologous repeated 6 kDa domains that inhibit trypsin or chymotrypsin, often circularly permuted. For a catalog of sequences and discussion of structural relationships, see Barta et al., (2002) Trends in Genetics 18:600-603. Sequences have been compiled in a database accessible at http://www.ba.itb.cnr.it/Plant-PIs (see also DeLeo, F. et al., (2002) Nucl. Acid Res. 30:347-348.)
In addition to the two-domain 12 kDa inhibitors, potatoes also contain lower levels of a series of single-domain inhibitors of approximately 6 kDa [Hass, G M, et al. (1982) Biochemistry 21:752-756] which are identical in sequence to the central portion of the two-domain proteins and are likely to be proteolytic products [Sanchez-Serrano supra]. Similar single-domain proteinase inhibitors (PI's) have been isolated from eggplant [Richardson, M. (1979) FEBS. Lett. 104:322-326] and tobacco [Pearce, G. et al. (1993) Plant Physiol. 102:639-644], although it is not known if they are derived from a larger precursor molecule. Both tomato and tobacco contain a gene encoding a three-domain inhibitor [Taylor, B H, et al. (1993) Plant Mol. Biol. 23:10 05-1014; Baladin, R. et al. (1995) Plant Mol. Biol. 27:1197-1204], and a gene encoding a six-domain inhibitor (NaPI-ii) has been isolated from the reproductive tissues of the ornamental tobacco, Nicotiana alata [Atkinson, A H, et al. (1993) Plant Cell 5:203-213].
NaPI-ii (SEQ ID NO:1) encodes a 40.3 kDa precursor protein that contains six inhibitory domains, two reactive against chymotrypsin and four reactive against trypsin [Atkinson supra]. Proteolytic processing of the precursor protein occurs in a linker region between domains resulting in the release of six mature, active inhibitors [Heath, R L, et al. (1995) Eur. J. Biochem. 230:250-257; Lee, M C S, et al. (1999) Nature Struct. Biol. 6:526-530]. In addition to the proteinase inhibitory domains, the precursor also has an N-terminal putative ER signal peptide and a C-terminal non-repeated domain which probably functions as a vacuolar sorting signal [Miller, E A, et al, (1999) Plant Cell 11:1499-1508; Nielsen, K J, et al. (1996) Biochemistry 35:369-378]. Previously we have shown that immature stigmas express two mRNAs that hybridise to the NaPI-ii cDNA [Atkinson supra]. One message of 1.4 kb corresponds to the six-domain inhibitor, while a second message of approximately 1.0 kb encodes a smaller isoform.
A second type two PI proteinase precursor having four repeated proteinase inhibitor domains has been isolated from N. alata stigmas, designated NaPI-iv, [Miller, E A, et al. (2000) Plant Mol. Biol. 42:329-333] (SEQ ID NO:2). The amino acid sequences of NaPI-ii and NaPI-iv align to reveal a high level of identity between the two proteins. (See
A nucleotide sequence of cDNA encoding NaPI-ii has been disclosed in PCT Publication No. WO 94/138810, SEQ ID NO:1 thereof, the entire publication incorporated herein by reference, to the extent not inconsistent herewith. The NaPI-iv cDNA sequence SEQ ID NO:2, GenBank Accession No. AF105340, is essentially that of NaPI-ii except for two alterations that result in the two conservative amino acid changes shown in
Expression of both NaPI-ii and NaPI-iv results in a protein which is post-translationally processed to yield individual mature 6 kDa proteinase inhibitor (PI) proteins having the designated trypsin (T) or chymotrypsin (C) inhibitory activities. Post-translational glycosylation has not been observed following expression in plant cells. Unprocessed precursor PI's retain the CTPP and are located outside the vacuole of the cell. Once the precursor protein is deposited in the vacuole, the C-terminal domain is rapidly removed and processing that yields individual 6 kDa PI's occurs [Miller (1999) supra].
The NaPI-ii precursor PI has been shown to adopt a circular structure by formation of disulfide bonds between the cys residues in the C2N (SEQ ID NO:1 or 2, amino acids 31-53) and C2C (SEQ ID NO:1, amino acids 344-373, SEQ ID NO:2, amino acids 228-2587) domains, [Lee (1999) supra]. The resulting product of cyclization of the precursor followed by post-translational proteolysis is a unique heterodimeric PI having chymotrypsin-inhibitor activity (C2).
Like other members of the type two family, the N. alata PI's inhibit the digestive proteases of several insect species [Heath, R L, et al. (1997) J. Insect Physiol. 43:833-842] and probably function to limit damage to floral tissues and leaves by insect pests. The PI's significantly retard the growth and development of Helicoverpa punctigera larvae when incorporated into artificial diets or expressed in the leaves of transgenic tobacco [Heath (1997) supra].
Various strategies have been adopted for expressing more than one transgene in a single transgenic plant. One technique has been to transform individual parent plants each with a single transgene and then to combine the transgenes in a single plant by crossing the parents, [Zhu, Q. et al. (1994) Bio/Technology 12:807-812; Bizily, S P, et al. (2000) Nat. Biotechnol. 18:213-217]. The breeding can be complicated where individual transgenes are recombined at different loci. The method is not applicable for vegetatively propagated plants.
Sequential single gene transformations have been carried out but have limited practical value because of limited availability of selectable markers for each transformation step.
The use of multiple transgenes linked on the same vector each separately controlled by its own copy of the same promoter has resulted in unexpected transcriptional silencing. [Matzke, A J M, et al. (1998) Curr. Opin. Plant Biol. 1:142-148] or non-uniform expression [Van der Elzen, P J M, et al. (1993) Phil. Trans. R. Soc. Land. B 342:271-278]. The use of different individual promoters to drive multiple linked transgenes appears feasible but expression is presumably subject to individual characteristics of each promoter.
Several investigators have reported adaptation of virus systems for expressing a polyprotein followed by specific protease cleavage in cis to release individual proteins. (See, e.g. Marcos, J F, et al. (1994) Plant Mol. Biol. 24:495-503; Beck von Bodman, S. et al. (1995) Bio/technology 13:587-591). The systems require introducing a viral protease to cleave the polyprotein with the possibility of undesired side effects of the introduced protease.
Urwin, P E, et al. (1998) Planta 204:472-479 described a dual proteinase inhibitor construct joined by a protease-sensitive propeptide from Pisum sativum, expressed in Arabidopsis. Only partial cleavage of the expressed polyprotein was reported. Using a 20 amino acid long linkage sequence, termed 2A, from foot-and-mouth disease virus, Halpin, C. et al. (1999) Plant J. 17:453-459 described constructing a polyprotein having two reporter coding regions joined by 2A in a single open reading frame. The 2A linker was reported to mediate co-translational cleavage at its own carboxy terminus by an enzyme-independent reaction. Although expression of the polyprotein and cleavage did occur, one of the resulting protein products retained 19 amino acids of the 2A linker and the 20th was attached to the other protein.
A similar result was described by Francois, I E J A, et al. (2002) Plant Physiol. 28:1346-1358, who joined coding regions of two proteins, DmAMPI, a plant defensin from seeds of Dahlia mercki1 and RsAFP2, a defensin from Raphanus sativus, using a propeptide of 16 amino acids from seeds of Impatiens balsamina. The propeptide of I. balsamina was obtained from a polyprotein precursor, IbAMP, described by Tailor, R A, et al. (1997) J. Biol. Chem. 272:24480-24487. The described polyprotein construct of DmAMPI and RsAFP2 was expressed and post-translationally cleaved in Arabidopsis; however, portions of the linking propeptide were found attached to the C- and N-termini of the linked proteins, regardless of their orientation in the polyprotein construct relative to the linker.
Using a composite linker of 29 amino-acids in length, Francois, I. F. I. A. et al. (2004) Plant Science 166:113-121 reported expression in Arabidopsis of DmAMP1 and RsAFP2 as a polyprotein precursor. The precursor was processed to yield DmAMP1 cross-reactive protein primarily in intracellular extracts and RsAFP2 cross-reactive protein primarily in extracellular fluid. The linker sequence was a composite of part of the I. balsamina linker and part of the foot-and-mouth disease 2A linker sequence. A recombination-based system for introducing a plurality of genes into a plant cell has been described by Chen, Q.-J., et al. (2006) Plant Mol. Biol. 62:927-936. Each gene has its own promoter and terminator.
Described herein is a multi-gene expression vehicle (MGEV) for concurrently expressing a plurality of genes in a plant cell, tissue or whole plant, under control of a single promoter. A MGEV can be constructed to express a linear polyprotein that lacks features necessary to cause the C-terminal and N-terminal ends to join together. The MGEV includes a single isolated polynucleotide whose sequence includes the following segments described by the function encoded by each segment: from 2 to 8 open reading frames (D2-8), each of which encodes a functional protein, and a plurality of linker segments (L1-7), each one situated between two D segments. The MGEV preferably includes, in addition, a 5′ terminal segment encoding an endoplasmic reticulum signal sequence (S) and a 3′-terminal segment encoding a C-terminal vacuole targeting peptide (V). Translation of a linear MGEV yields a linear polyprotein which is further processed by cleavage at the linker (L) segments, to separate the protein domains from one another. Optionally, in its circular form, the MGEV additionally includes segments encoding a first “C1asp” peptide (C2N) on the C-terminal side of S and a second “C1asp” peptide (C2C) on the N-terminal side of V. Preferably, the C2N and C2C proteins have secondary and tertiary structures that allow them to interact to form a hetero-dimer that can be covalently linked together by post-translational formation of disulfide bonds, thereby forming a “circular” polyprotein (having a cyclic topology). In one embodiment, the cross-linked C2N-C2C dimer has activity as a chymotrypsin inhibitor (C2). The circular MGEV can have from 3-8 reading frames (D3-8) with linkers between each domain and each “clasp” peptide (L4-8). Ultimately, the circular polyprotein is also cleaved at each L segment. In both linear and circular forms, the signal polypeptide (S) and the vacuole targeting peptide (V) function to control intracellular transport of the entire polyprotein, prior to cleavage at L sites.
The following abbreviations are used in all plasmid maps herein (
Various MGEV structures are detailed herein and in the following examples. A general MGEV structure encoding a circular polyprotein (MGEV-P) is diagrammed as follows:
S-C2N-(LjDk)m-LjC2C-V
where each capital letter symbolizes a polynucleotide encoding a segment of amino acids designated according to its function, thus: S is a polynucleotide segment with an open reading frame encoding a signal peptide; Dk is a polynucleotide segment with an open reading frame encoding a functional protein (hereinafter a “Domain”) wherein k represents an ordinal number to identify any single functional Domain selected from a group of domains having from 3 to m members and at least one of D does not encode a type two protease inhibitor; Lj is a polynucleotide segment with an open reading frame encoding a linker polypeptide where Lj is a ordinal number to identify each single linker (L) selected from a group having from 3 to m+1 members; C2N is a polynucleotide segment with an open reading frame encoding a N-terminal clasp peptide; C2C is a polynucleotide with an open reading frame encoding a C-terminal clasp peptide; V is a vacuolar targeting peptide; m is a cardinal number from 3-8; and S, C2N, L, D, C2C and V are all in the same reading frame same as each other. As an example, a MGEV encoding 3 functional domains (D) can be diagrammed as shown above, where m is 3, k is 1, 2 or 3, j is 1, 2, 3, or 4. In another linear embodiment, described below, clasp proteins are omitted or truncated. In the absence of a clasp peptide, there is no requirement for any of D to encode a type two proteinase inhibitor.
Lj encodes a linker amino acid sequence as described herein. Each Lj can have the same or a different sequence. A generic linker amino acid sequence is given at SEQ ID NO:17.
In a plant cell, the MGEV encoded protein (MGEV-P) undergoes several steps of post-translational processing. These include intracellular transport to the endoplasmic reticulum, provided the leader (S) is present, followed by removal of S and subsequent transport to an intracellular storage vacuole provided the vacuolar targeting sequence (V) is present. V is removed in the vacuole. If C2N and C2C are present, the ends of the MGEV-P become joined together to form a closed loop, diagrammed as follows:
where C2N, L1-4, D1-3, C2C, and V are as described supra.
Post-translational proteolysis cleavage at each linker and between C2C and V results in release of D1, D2, D3 and, in one embodiment, C2, as separate proteins. Expression of the MGEV thereby results in concurrent expression of at least three separate proteins at least one of which is not a type two proteinase inhibitor, from a single promoter.
A circular MGEV can encode from 3 to 8 functional domains (D), concurrently expressed. Concurrent expression is defined herein to mean the intracellular synthesis of a plurality of functional proteins from a single transcript. Concurrent expression is especially useful when it is desired or necessary to produce and accumulate large amounts of proteins in a plant cell, for example, plant protectant proteins, or economically significant proteins, or when it is advantageous to control the relative amounts of expressed proteins, or for expression of certain proteins, such as cysteine-rich peptides, that are normally expressed poorly in plant cells. When the MGEV includes a vacuole targeting peptide (V), the concurrently expressed proteins are accumulated in a storage vacuole in the cell, which can serve two purposes: (1) to provide the proteins in concentrated form to maintain an effective dose of plant protectant in the event of pathogen attack, or to ease purification of an economically valuable protein; and (2) to sequester otherwise toxic proteins which can confer added pest resistance and economic value to a plant expressing such proteins. V can be combined with any domain to be expressed, most conveniently at the 3′-end of MGEV. More than one V can be included if desired. In the absence of V, proteins released from MGEV-P by proteolysis can be exported from the cell.
The expressed components of an MGEV are described herein in greater detail.
The protein domains (D) encoded by open reading frames of the MGEV nucleotide sequence can, in principle, be any protein. No upper size limit is known for a protein expressible as a component of a MGEV. Exemplified herein are data demonstrating concurrent expression of individual domains encoding proteins ranging from about 5 kDa to greater than 65 kDa. Practical considerations known to those skilled in the art can be considered when choosing proteins appropriate for expression using an MGEV. For example, very large proteins may be expressed individually more efficiently, rather than as part of a MGEV. Certain proteins may sterically interfere with cyclization under certain circumstances. Each protein domain (D) is connected to a linker peptide (L) by peptide bonds at the N-terminal and C-terminal amino acids of the domain.
It is presently believed that efficiency of post-translational peptide cleavage that liberates individual protein domains from the MGEV-PIs maximized when the N- and C-termini of each domain and connecting linkers are exposed by the protein conformation to the aqueous environment on the surface of the protein, rather than sequestered internally within the protein. Therefore, candidate proteins for expression as part of the MGEV-P preferably have exposed N- and C-terminal amino acids.
Examples of proteins which can be expressed using an MGEV include (without limitation) potato type one PI's, potato type two PI's, plant defensins, animal defensins, proteinaceous toxins, chimeric and fusion proteins, as well as indicator proteins such as Green Fluorescent Protein (GFP), 28 kDa, and beta-glucuronidase (GUS), 68 kDa. Examples of protein-coding domains that can be expressed in the MGEV include plant protection proteins such as potato proteinase inhibitors of type one (Pot 1A), plant seed defensins, plant floral defensins, insect-toxic peptides such as scorpion toxin, Bacillus thuringiensis toxins, heat shock proteins, Bowman-Birk trypsin inhibitors, and cystatins and indicators such as green fluorescent protein (GFP) and beta-glucuronidase (GUS). Proteins of economic value for purposes other than plant protection can be expressed using the MGEV, taking advantage of high expression levels, including anti-microbial peptides. antibody fragments and the like suitable for medical use. Also large hetero-dimeric or hetero-multimeric proteins are especially suitable for MGEV expression where concurrent and correctly proportional expression is desired. At least one protein encoded by a MGEV is not a type two PI. The MGEV is particularly useful for expression of proteins that may be toxic to the cell in which they are expressed, by providing for transport to, and sequestration in, a storage vacuole within the plant cell.
A linker (L) is a short peptide positioned between each domain that separates each adjacent domain and exposes a peptidase-sensitive site for post-translational cleavage between individual domains. The amino acid sequence—EEKKN (SEQ ID NO:5)—is an example of a linker peptide. Other amino acid sequences can serve as linkers, for example, sequences where E and K are substituted by similar amino acids, such as D (asp) or R (arg) or N (asn) is substituted by a Q (gin). A consensus linker sequence can be expressed as X1X2X3X4X5 where X1 is E (glu) or D (asp), X2 is E (glu) or D (asp), X3 is K (lys) or R (arg), X4 is K (lys) or R (arg) and X5 is N (asn) or Q (gln) (SEQ ID NO:17). The linker provides a highly hydrophilic segment that exposes a proteolytic cleavage site (N-X) to the outer surface of MGEV-P. Any short highly hydrophilic peptide can serve as a linker in the MGEV-P. The linker peptides described herein are advantageous because post-translational processing of domains joined by a linker can result in removal of the entire linker in transgenic plants. (See Heath, R. L. et al., (1995) Eur. J. Biochem. 230:250-257).
The leader peptide, also referred to as a signal peptide (S), is a sequence of about 10 to about 30 mostly hydrophobic amino acids which serves a transport function for intracellular transport. Many signal peptides are known in the art. Any known signal peptide can be used in the MGEV-P, as well as modifications thereof wherein homologous amino acids are substituted.
The vacuole targeting peptide (V) is located at the C-terminus of the MGEV-P. A variety of vacuolar targeting determinants are known to exist in plant cells, see, e.g. Maruyama et al. Plant Cell (2006) 18:1253-1273. Suitable vacuolar targeting peptides can be chosen from a wide variety of known candidates. Also, a suitable V segment need not be placed at the C-terminus of the MGEV, but could, in principle be located elsewhere in the sequence; for example attached to the N-terminus of C2N, between S and C2N. In one embodiment, a suitable sequence can be one which binds to the known BP-80 vacuolar sorting receptor. Any such vacuole targeting sequence that binds BP-80 or a homolog thereof can be used as a component of the MGEV-P. Another example of a suitable vacuole targeting sequence is shown in Miller, et al. supra,
The clasp segments, C2N and C2C are represented herein by amino acids 30-48 (C2N) and 228-257 (C2C) SEQ ID NO:6. The folded configuration of peptides C2N and C2C is such that they readily bind to one another, and the heterodimer formed by the binding is then stabilized covalently by formation of inter-peptide disulfide cross-links. The cross-linked [C2N:C2C] protein has chymotrypsin activity and is designated simply as C2 herein. In the MGEV-P structure, formation of C2 results in cyclization of MGEV-P with a C-terminal extension, the vacuole targeting peptide, V. A clasp structure can be formed using any of the type 2 inhibitors regardless of protease specificity, because of the high degree of homology among them. Deletion of the four amino acid sequence PRNP (or PKNP in the case of T5) which is common to these inhibitors will create the appropriate N-terminal and C-terminal segments of a clasp peptide. Formation of a cyclic structure is not necessary for activity of MGEV-P. A cyclic structure of MGEV-PIs considered advantageous for efficient intracellular transport. A further advantage of the cyclic configuration is that the additional inhibitor thereby formed is a useful plant protectant against insect damage.
The total or partial deletion of C2N and C2C can prevent formation of a cyclic structure and result in a linear configuration. The invention includes both linear and cyclic configurations of MGEV-P. A linear MGEV is advantageous whenever a large protein, a mix of large and small proteins, or a protein lacking a compact tertiary structure is to be expressed. In certain circumstances expression levels can be increased by use of a linear MGEV-P instead of the cyclic form. Targeting to the endoplasmic reticulum by S and vacuolar targeting by V can occur as previously described. A linear MGEV can have as few as two domains. Post-translational processing of linear MGEV-P can occur as described, with release of individual active domains (Dk). A diagram of a linear MGEV-P having 3 protein domains lacking C2N and C2C is shown, wherein non-specific peptides PN and PC are provided in place of C2N and C2C, respectively.
S-PN-(LjDk)mLjPC-V
where j is 1, 2 or 4, k is 1, 2 or 3, m is 3.
PN and PC can be modified or partially deleted versions of C2N and C2C, respectively. Preferably, C2N and C2C are entirely deleted, such that a linear 3-domain MGEV has the diagram structure:
S-(DkLj)mDk+1V
where j and k are 1 or 2 and m is 2.
As noted previously, V need not be at the C-terminus, but could be located elsewhere in the sequence, for example between S and D.
The linear MGEV-P can have up to eight functional protein domains, at least one of which is not a type two proteinase inhibitor. As with cyclic MGEV-P, the linear form can be exported from the cell by deletion of the vacuole targeting sequence, V.
Constructing a MGEV can be carried out by known methods of combining the nucleic acid segments in the designated order, by DNA synthesis, or a combination of both methods. A convenient method is to employ components of naturally-occurring type two PI multimers, such as NaPI-iv from N. alata, SEQ ID NO:2 [Miller, (2000) supra, GenBank accession number AF105340]. One or more open reading frames encoding a functional protein domain of interest that is not a type two PI can be inserted together with appropriate linkers into the naturally-occurring multimer, thereby increasing the number of expressed domains, or pre-existing domains can be deleted, followed by insertion of desired domain-coding segments to keep the total number of domains unchanged as long as all coding segments remain in the same reading frame from one to the next. Examples of protein-coding domains that can be expressed in the MGEV include plant protection proteins such as potato proteinase inhibitors of type one, for example as disclosed in International Publication No. WO 2004/094630, including PotIA exemplified herein, plant seed defensins, plant floral defensins, insect-toxic peptides such as scorpion toxin, Bacillus thuringiensis toxins, heat shock proteins, Bowman-Birk trypsin inhibitors, and cystatins and indicators such as green fluorescent protein (GFP) and beta-glucuronidase (GUS). Proteins of economic value for purposes other than plant protection can be expressed using the MGEV, taking advantage of high expression levels, including anti-microbial peptides. antibody fragments and the like suitable for medical use. Also large hetero-dimeric or hetero-multimeric proteins are especially suitable for MGEV expression where concurrent and correctly proportional expression is desired.
The following Examples demonstrate construction of MGEV's encoding plant-protective proteins, plant transformation with MGEV, transgenic plants containing and expressing the MGEV and protection from plant pests due to expression of non-Potato Type Two proteins encoded within a MGEV, and MGEVs encoding a mix of large and small proteins. These Examples are presented to illustrate, but not limit, the invention as claimed.
A MGEV can be expressed in plants or plant cells after being incorporated into a plant transformation vector. Many plant transformation vectors are well known and available to those skilled in the art, e.g., BIN19 (Bevan, (1984) Nucl. Acid Res. 12:8711-8721), pBI 121 (Chen, P-Y, et al., (2003) Molecular Breeding 11:287-293), PHEX 22 (U.S. Pat. No. 7,041,877), and vectors exemplified herein. Such vectors are well-known in the art, often termed “binary” vectors from their ability to replicate in a bacteria such as Agrobacterim tumefaciens and in a plant cell. A typical plant transformation vector, such as exemplified herein, includes genetic elements for expressing a selectable marker such as NPTII under control of a suitable promoter and terminator sequences, active in the plant cells to be transformed (hereinafter “plant-active” promoter or terminator) a site for inserting a gene of interest, including a MGEV under expression control of suitable plant-active promoter and plant-active terminator sequences and T-DNA borders flanking the MGEV and selectable marker to provide integration of the genes into the plant genome.
Plants are transformed using a strain of A. tumefaciens, typically strain LBA4404 which is widely available. After constructing a plant transformation vector that carries a MGEV encoding the desired proteins, the vector is used to transform an A. tumefaciens strain such as LBA4404. The transformed LBA4404 is then used to transform the desired plant cells using an art-known protocol appropriate for the plant species to be transformed. Standard and art-recognized protocols for selecting transformed plant cells, multiplication and regeneration of selected cells are employed to obtain transgenic plants. The examples herein further disclose methods and materials used for transformation and regeneration of cotton plants, as well as transgenic cotton plants transformed by and expressing a variety of MGEVs. A MGEV can be transferred into plant cells by any of several known methods besides those exemplified herein. Examples of well-known methods include microprojectile bombardment, electroporation, and other biological vectors including other bacteria or viruses.
The MGEV can be used for multigene expression in any monocotylodenous or dicotyledonous plant. Particularly, useful plants are food crops such as corn (maize) wheat, rice, barley, soybean and sugarcane and oilseed crops such as sunflower and rape. Particularly useful non-food common crops include cotton, flax and other fiber crops. Flower and ornamental crops include rose, carnation, petunia, lisianthus, lily, iris, tulip, freesia, delphinium, limonium and pelargonium.
Techniques for introducing vectors, chimeric genetic constructs and the like into cells include, but are not limited to, transformation using CaCl2 and variations thereof, direct DNA uptake into protoplasts, PEG-mediated uptake to protoplasts, microparticle bombardment, electroporation, microinjection of DNA, microparticle bombardment of tissue explants or cells, vacuum-infiltration of tissue with nucleic acid, and T-DNA-mediated transfer from Agrobacterium to the plant tissue.
For microparticle bombardment of cells, a microparticle is propelled into a cell to produce a transformed cell. Any suitable ballistic cell transformation methodology and apparatus can be used in performing the present invention. Exemplary procedures are disclosed in Sanford and Wolf (U.S. Pat. Nos. 4,945,050, 5,036,006, 5,100,792, 5,371,015). When using ballistic transformation procedures, the genetic construct can incorporate a plasmid capable of replicating in the cell to be transformed.
Examples of microparticles suitable for use in such systems include 0.1 to 10 μm and more particularly 10.5 to 5 μm tungsten or gold spheres. The DNA construct can be deposited on the microparticle by any suitable technique, such as by precipitation.
Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, can be transformed with a MGEV of the present invention and a whole plant generated therefrom, as exemplified herein. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Examples of tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g. apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g. cotyledon meristem and hypocotyl meristem).
The regenerated transformed plants can be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed to give a homozygous second generation (or T2) transformant and the T2 plants further propagated through classical breeding techniques.
Accordingly, this aspect of the present invention, insofar as it relates to plants, further extends to progeny of the plants engineered to express the nucleic acid of the MGEV as well as vegetative, propagative and reproductive parts of the plants, such as flowers (including cut or severed flowers), parts of plants, fibrous material from plants (for example, cotton) and reproductive portions including cuttings, pollen, seeds and callus.
Another aspect of the present invention provides a genetically modified plant cell or multicellular plant or progeny thereof or parts of a genetically modified plant capable of producing a protein or peptide encoded by the MGEV as herein described wherein said transgenic plant has acquired a new phenotypic trait associated with expression of the protein or peptide.
MGEV structures and MGEV expression vectors exemplified herein are listed in Table 2, together with the number of the Example where they are described. Sequence ID listings are listed in Table 3.
The MGEV described in this example (MGEV-5) SEQ ID NO:6 has the structure diagrammed as:
S-C2N-L1D1-L2D2-L3D3-L4-C2C-V;
wherein L1-4 encodes the linker amino acid sequence -EEKKN—SEQ ID NO:5, D1 encodes a potato type two trypsin inhibitor, T1 SEQ ID NO:3; SEQ ID NO:1 amino acids 112-164; D2 encodes a potato type one chymotrypsin inhibitor, potato Pot 1A SEQ ID NO:11, (also SEQ ID NO:5, bases 352-376); D3 encodes a Type Two chymotrypsin inhibitor, C1 SEQ ID NO:2 amino acids 54-106; C2N SEQ ID NO:1 amino acids 31-48 and C2C SEQ ID NO:1 amino acids 344-373 encode peptides that interact with each other to form a heterodimer C2 stabilized by disulfide crosslinks, the cross-linked protein having potato type two chymotrypsin inhibitor activity. S encodes a signal peptide and V encodes a vacuole translocation peptide.
Amino acid sequences encoded by the above-identified segments are described in the following sources:
A multipurpose vector, pRR19 was constructed. The vector contained sequences obtained from NaPI-iv SEQ ID NO:2 and NaPI-ii SEQ ID NO: 1 [Miller (2000) supra] plus restriction sites for insertion of new genes. The entire MGEV-1 sequence was assembled in consecutive order into pRR19.
The vector pRR19 was designed to allow convenient modular assembly of linkers (L) and open reading frames (D) into a MGEV having the desired combination of components. As step 1, polymerase chain reaction (PCR) was used to amplify the respective N- and C-terminal end segments of NaPI-iv, specifically S-C2N (SEQ ID NO:2 amino acids 1-48) and C2C-V (SEQ ID NO:2 amino acids 228-281), and to provide Xho I restriction sites. The Xho I restriction sites were provided to permit joining of desired segments between the terminal segments, such that the amplified segments had the diagram structure S-C2NL1-XhoI and XhoI-C2C-V, respectively. After cutting and ligation, the segment S-C2N-L1-Xho1-C2C-V was cloned into the PGEM T-Easy (Promega, Madison, Wis.) vector.
Any desired DNA segment having Xho1 sites at its N and C termini could then be inserted into the XhoI site of the resulting vector.
As the step 2, in parallel preparations, DNA encoding the T1 of NaPI-ii (SEQ ID NO:1, amino acids 112-164) (to be in position D1 in MGEV-5) and the DNA encoding the C1 domain of NaPI-iv (SEQ ID NO:2, amino acids 54-106) (to be position D3 in MGEV-5) were PCR-amplified with restriction sites added as diagrammed:
Xho1-T1-L1—Xba1, and Xba1-C1-L1-Xho1.
Each of the constructs was separately cloned into PGEM T-easy vectors, digested with Xba1 and Xho1 and purified.
The modified T1 and C1 domains from the preceding step were combined in a DNA ligation reaction mixture with Xho1-digested product of the first step. The ligation mixture was transformed into E. coli XL1-Blue cells (Stratagene, LaJolla, Calif.) and restriction digests and sequencing were carried out to confirm the desired orientation of and order of the proteinase inhibitor domains. The predicted ligation reactions were DNA segments encoding the following components:
The ligation product, as verified by electrophoresis of restriction digests and sequence analysis, was
S-C2N-L1-Xho1-T1-L1-Xba1-C1-L1-Xho1-C2C-V
The ligation product contained a unique Xba1 site (underlined) into which could be inserted any desired coding sequence provided with Xba1 restriction sites at both ends. The vector having the described construct was designated pRR19.
For the D2 domain, the DNA coding for Pot 1A, previously described, was provided with a linker (L) at the C-terminal-coding end, followed by Xba1 restriction sites at the 3′ and 5′ ends. Insertion at the Xba1 site of pRR19 resulted in a construct that was then inserted into pAM9 (pAM9 was modified from PDHA, Tabe et al., Journal of Animal Science, 73: 2752-2759, 1995) to produce MGEV-5. Insertion in pAM9 resulted in the attachment of the 35S CaMV promoter at the 3′ end and the 35S CaMV terminator at the 5′ end. MGEV-5 was then inserted into pBIN19 at the EcoRI site resulting in vector PHEX 29, diagrammed in
The use of restriction sites in MGEV-5 could be avoided, if desired, by using DNA synthesis to make the disclosed MGEV-5 sequence of Table 1. See also SEQ ID NO:6 (DNA sequence) and SEQ ID NO:12 (deduced amino acid sequence).
Seeds of Gossyipium hirsutum cultivar Coker 315 were surface sterilized in sodium hypochlorite (2% available chlorine) for 60 min followed by several washes in sterile water. The sterilized seed were sown onto Cotton Seed Medium (CSM) [0.22% w/v MS (Murashige and Skoog salt mixture Austratec M524), 0.05% w/v B5 vitamins (Sigma G1019), 1.5% w/v glucose (Austratec G386), 0.2% w/v gellan gum Gelrite, trademark of Merck & Co., (Phyto Technology Laboratories), pH 5,8] and incubated at 30° C. in the dark for 10 days. A. tumefaciens (LBA4404) transformed with the pHEX29 construct was grown overnight in 25 ml LB medium supplemented with the antibiotic kanamycin (50 μg/mL) at 28° C. The absorbance at 550 nm was measured and the cells were diluted to 2×108 cells per ml in MS liquid media (0.43% w/v Murashige and Skoog basal salts, pH 5.8). Cotton hypocotyls were cut into 1.5-2 cm pieces and mixed briefly (0.5-3 min) in the diluted Agrobacterium culture. The explants were drained and transferred to medium 1 (0.43% w/v Murashige and Skoog salt mixture, 0.1% v/v Gamborg's B5 vitamin solution (Sigma), 0.1 g/L myo-inositol, 0.9 g/L MgCl2, (hexahydrate), 1.9 g/L potassium nitrate, 0.2% w/v Gelrite, 3% w/v glucose, pH 5.8) overlayed with sterile filter paper and incubated for 3 days at 26° C. under lights.
Following co-cultivation, explants were transferred to medium 2 (medium 1 plus 0.1 mg/L kinetin, 0.1 mg/L 2,4-D, 500 mg/L carbenicillin, 35 mg/L kanamycin) and maintained at 30° C. under low light. After 4 weeks explants were transferred to medium 3 (medium 1 plus 500 mg/L carbenicillin, 25 mg/L kanamycin) and maintained at 30° C. under low light. Explants and callus were sub-cultured every 4 weeks on medium 3 and maintained at 30° C. under low light. Embryos were excised from the tissue and germinated in medium 4 (1.2 mM CaCl22H2O, 5.0 mM KNO3, 2.0 mM MgSO47H2O, 3.0 mM NH4NO3, 0.2 mM KH2PO4, 4 μM nicotinic acid, 4 μM pyridoxine HCl, 4 μM thiamine HCl, 30 μM H3BO3, 30 μM MnSO4H2O, 9 μM ZnSO47H2O, 1.5 μM KI, 0.9 μM Na2MoO42H2O, 0.03 μM CuSO45H2O, 0.03 μM CoCl26H2O, 15 μM FeNaEDTA, 0.5% w/v glucose, 0.3% w/v gellan gum Gelrite, pH 5.5) and maintained at 30° C. under high light.
Germinated embryos were then transferred to Magenta boxes containing medium 4 and maintained at 30° C. under high light. Once a plant has formed a good root system and produced several new leaves it was transferred to soil in pots and acclimatised in a growth cabinet at 28° C. and then grown in a glasshouse at (27-29° C. day, 20-24° C. night).
PCR Analysis
DNA isolation: Cotton leaf discs (0.5-0.7 cm) were sampled from the 2nd fully expanded leaf, avoiding vein tissue. Extraction solution (100 μl) from the REDExtract-N-Amp Plant PCR kit (Sigma) was added to each leaf disc ensuring the tissue was fully submerged. Samples were heated at 95° C. on a heat block for 10 minutes before vortexing. Dilution solution (100 μl, Sigma) was added and the sample was vortexed thoroughly and placed on ice.
The PCR reaction mix consisted of the following components: 10 μl PCR ready mix (REDExtract-N-Amp, Sigma) 0.8 μl forward primer, 0.8 μl reverse primer, 2.8 μl H2O, 4 μl DNA extract (from above). PCR conditions were 94° C., 4 min, followed by 33 cycles of 94° C. 30 sec, 62° C. 30 sec, 72° C. 1 min followed by 72° C. for 10 min. Samples were stored at 4° C.
Primers:
Detection of NaPI and StPot1A in Transgenic Cotton
ELISA
Protein extract: leaves were excised from plants grown either in the growth cabinet or in the glasshouse. The tissue (100 mg) was frozen in liquid nitrogen and ground in a mixer mill (Retsch MM300) for 2×15 sec at frequency 30. 1 mL of 2% insoluble PVP (Polyclar)/PBS/0.05% Tween 20 was added prior to vortexing for 20 sec. The samples were centrifuged for 10 min and the supernatant was collected.
Coat ELISA plate (Nunc Maxisorp #442404) with 100 μL/well of primary antibody in PBS.
100 ng/well of anti-NaPI (polyclonal antibody was made by a standard method to purified NaPI peptides isolated from stigmas) or anti-Pot 1A (antibody made to Pot1A that was expressed as a dimer with C1 in E. coli and then cleaved and separately purified), Incubate overnight at 4° C. in a humid box. Wash plates 2 min×4 with PBS/0.05% Tween 20. Block plate with 200 μL/well 3% BSA (Sigma A-7030: 98% ELISA grade) in PBS. Incubate for 2 hr at 25° C. Wash plates 2 min×4 with PBS/0.05% Tween 20. The anti-NaPI antibody binds to the T and C protease inhibitors of N. alata.
Apply 100 μL/well of cotton protein extracts (diluted in PBS/0.05% Tween 20). Incubate 2 hr at 25° C. Wash plates 2 min×4 with PBS/0.05% Tween 20. Apply 100 μL/well of secondary antibody in PBS (50 ng/well biotin-labelled NaPI antibody, 200 ng/well biotin-labelled Pot 1A antibody). Incubate for 1 hr at 25° C. The biotin labelled antibody is prepared using the EZ-link Sulfo-NHS-LC-biotinylation kit (Pierce). Use 2 ml of protein A purified antibody and 2 mg of the biotin reagent.
Wash plates 2 min×4 with PBS/0.05% Tween 20. Apply 100 μL/well NeutriAvidin HRP-conjugate (Pierce #31001; 1:1000 dilution; 0.1 μL/well) in PBS. Incubate for 1 hr at 25° C.
Wash plates 2 min×4 with PBS/0.05% Tween 20, followed by 2 min×2 with H2O. Just before use, prepare substrate by dissolving 1 ImmunoPure OPD tablet (Pierce #34006) in 9 mL H2O, then add 1 mL stable peroxide buffer (10×, Pierce #34062). Add 100 μL/well substrate. Incubate at 25° C. until colour develops. Stop reaction with 50 μL 2.5 M sulfuric acid. Measure absorbance at 490 nm in plate reader (Molecular Devices, Milenia Kinetic Analyzer).
Immunoblot Analysis
Leaves were excised from plants grown either in the growth cabinet or in the glasshouse. Leaf tissue (100 mg) was frozen in liquid nitrogen and ground to a fine powder in a mixer mill (Retsch MM300), for 2×15 sec at frequency 30. The powder was added to 2× sample buffer (300 μl, Novex NuPAGE LDS sample buffer, 10% v/v β-mercaptoethanol), vortexed for 30 sec, boiled for 5 min and then centrifuged at 14,000 rpm for 10 min and the supernatant retained for SDS-PAGE. Alternatively, the powder was added to 1 ml acetone, vortexed thoroughly and centrifuged at 14,000 rpm (18,000 g) for 2 min and the supernatent discarded. The pellet was resuspended in 300 μl of IP lysis buffer (50 mM Tris pH 8, 5 mM EDTA, 150 mM NaCl, 0.1% Triton X-100) with 2% Polyclar AT (water-soluble polyvinyl polypyrrolidine) by vortexing thoroughly and supernatant was collected after centrifugation at 14,000 rpm for 10 min. For analysis by SDS-PAGE, 30 μl of sample in 1× sample buffer (Novex NUPAGE LDS sample buffer) and 5% v/v β-mercaptoethanol was used.
Extracted leaf proteins were separated by SDS-PAGE on preformed 4-12% w/v polyacrylamide gradient gels (Novex, NuPAGE bis-tris, MES buffer) for 35 min at 200V in a Novex X Cell II mini-cell electrophoresis apparatus. Prestained molecular weight markers (Novex SeeBlue Plus 2) were included as a standard. Proteins were transferred to nitrocellulose membrane (Osmonics 0.22 micron NitroBind) for 60 min at 30V using the Novex X Cell mini-cell electrophoresis apparatus in NuPAGE transfer buffer with 10% v/v methanol. After transfer, membranes were incubated for 1 min in isopropanol, followed by a 5 min wash in TBS.
The membrane was blocked for 1 h in 3% w/v BSA at RT followed by incubation with primary antibody overnight at RT (NaPI antibody: 1:2000 dilution in TBS/1% BSA of 1 mg/ml stock, Pot 1A antibody: 1:1000 in TBS/1% BSA of 1 mg/ml stock). The membrane was washed 5×10 min in TBST before incubation with goat anti-rabbit IgG conjugated to horseradish peroxidase for 60 min at RT (Pierce, 1:100,000 dilution in TBS). Five further 10 min TBST washes were performed before the membrane was incubated with the SuperSignal West Pico Chemiluminescent substrate (Pierce) according to the Manufacturer's instructions. Membranes were exposed to ECL Hyperfilm (Amersham).
Results
From 2 experiments (CT 89 and CT 90) we produced 86 potential transgenic plants. All plants were screened by PCR using the npt primers and the StPotIA primers. Plants positive for npt 11 were assessed for NaPI protein expression by ELISA. 38 plants were expressing detectable levels of NaPI (
Line 89.5.1 was selfed and the T2 progeny seed grown and the plants assessed for NaPI expression by ELISA. 20 of the 27 plants (74%) were expressing NaPI and 7 plants (26%) were null segregants (
Immunoblot analysis of selected lines using the NaPI antibody confirmed that the precursor protein and the processed peptides were present (
The results demonstrate that a MGEV encoding four peptides, at least one of which is not a type 2 protease inhibitor, can be constructed using conventional methods and used to successfully transform a plant (cotton) of a different species than that from which any of the component DNA segments were derived. The encoded protein is expressed and post-translationally processed to yield component peptides of the expected size.
The MGEV described in this example (MGEV-8) has the structure diagrammed as:
S-D1L1D2L2D3L3-V
The Xho 1-flanked T1-Xba 1-C1 fragment was cut from the multipurpose vector pRR20 (see Example 3) and ligated into the S-Xho 1-V construct described above, resulting in a S-Xho 1-T1-Xba 1-C1-Xho 1-V construct. This linear multipurpose vector was designated pSP1.
The mature domain of potato Pot 1A (see Example 1) was PCR-amplified with an EEKKN linker sequence (SEQ ID NO: 5) at the 3′ end and with Xba 1 sites at both ends. This was then ligated into the Xba 1 site of pSP1 to produce MGEV-8 (
Transient Expression in Cotton Cotyledons
pHEX 56 was introduced into A. tumefaciens and the expression of T1, C1 and Pot 1A was determined by a transient assay with cotton cotyledons.
Bacterial “lawns” of the Agrobacterium were spread on selective plates and grown in the dark at 30° C. for 3 days. Bacteria were then resuspended to an OD600 of 1.0 in infiltration buffer (10 mM magnesium chloride and 10 μM acetosyringone (0.1 M stock in DMSO)) and incubated at room temperature for 2-4 h. Cotton plants were grown for 8 days in a controlled temperature growth cabinet (25° C., 16 h/8 h light/dark cycle). The underside of the cotyledons was infiltrated by gently pressing a 1 mL syringe against the leaf and filling the leaf cavity with the Agrobacterium suspension. The area of infiltration (indicated by darkening) was noted on the topside of the leaf. A maximum of 4 infiltrations were performed per cotyledon. Plants were grown for a further 4 days. The infiltrated areas were then cut out, weighed and frozen in liquid nitrogen. Protein expression was determined by ELISA and immunoblots as described in Example 1.
Results
NaPI (
The results confirm previous conclusions from Example 1 and demonstrate, in addition, expression of PotIA. The results also demonstrate that cyclization of a primary MGEV expression product is not required for processing to yield predicted component peptides.
Note: In Examples 3-16, linker peptides (L) are omitted from the MGEV diagram in order to simplify the diagram.
The MGEV described in this example (MGEV-6) has the structure diagrammed as:
(See also
MGEV-6, expressing a defensin and 3 potato type two PI's, was constructed essentially as described for MGEV-5 (Example 1) except that a modified multipurpose vector (pRR20) was used and a defensin coding sequence was inserted instead of Pot 1A. The defensin was NaD1 as described in U.S. Pat. No. 7,041,877, and herein SEQ ID NO:14, amino acids 26-72, having a mature defensin domain but lacking the C-terminal acidic peptide tail, and without the N-terminal signal peptide.
The modified multipurpose vector (pRR20) is the same as the multipurpose vector (pRR19) described in Example 1, except that the codon encoding N in the EEKKN linker (SEQ ID NO:5) (L1) of the Xho1-T1-L1-XbaI DNA fragment was changed from AAT to AAC SEQ ID NO:12. This deleted an undersired Eco R1 restriction site that was present in pRR19.
NaD1 DNA was ligated into the Xba 1 site of pRR20, then excised with Bam H1 and Sal 1 and the complete fragment inserted into pAM9 to produce MGEV-6. MGEV-6 was then inserted into pBIN19 to produce the vector pHEX31, diagrammed in
Transformation of Cotton
Cotton transformation with pHEX31 was carried out as described in Example 1.
Protein Detection
Protein expression was determined by ELISA as described in Example 1. The primary NaD1 antibody and the secondary NaD1-biotin antibody were used at 50 ng/well.
Immunoblot analysis was carried out as described in Example 1 with the modification described in Example 2. The primary NaD1 antibody was diluted 1:1,000 dilution from a 1 mg/ml stock and the secondary antibody (goat anti-rabbit IgG conjugated to horseradish peroxidase) was used at a 1:50,000 dilution.
Results
From one experiment (CT 93) 88 potential transgenic plants were produced. All plants were screened by PCR using the nptII primers and primers specific for NaD1. 57 plants were positive for the presence of the nptII gene, with 33 of these plants also carrying the NaD1 gene. PCR positive plants were assessed for NaPI and NaD1 protein expression by ELISA. 13 primary transgenic plants were expressing detectable levels of NaPI and NaD1.
Three transgenic lines (93.4, 93.36 and 93.279) were selected for further study. The primary transgenic lines were selfed and the T2 seed collected. T2 plants from two of these lines (93.4 and 93.279) were assessed for NaPI expression (
Immunoblot analysis of lines 93.4 and 93.36 using the NaPI antibody confirmed that the precursor protein and the processed peptides were present (
The results demonstrate utility of MGEV for simultaneously expressing a protein other than a protease inhibitor (NaD1, a defensin).
The MGEV described in this example (MGEV-7) has the structure diagrammed as:
(See Also
MGEV-7 has a similar structure to MGEV-5 (Example 1) except that a DNA sequence encoding a Green Fluorescent Protein (GFP) was inserted in place of Pot 1A. The GFP is a soluble, highly fluorescent variant of green fluorescent protein (GFP) for use in higher plants (Davies, S J and Vierstra, R D: Plant Mol. Biol. 36(4): 521-528 (1998). The DNA was obtained from TAIR (the Arabidopsis information resource) (SEQ ID NO:13). Sequence information is available from Genbank at accession number U70495, and herein at SEQ ID NO:13.
For construction of MGEV-7, a third multipurpose vector (pRR21) was used. This was made in the same way as pRR20 except that the DNA encoding the C1 domain of NaPI-iv was PCR-amplified with an extra EEKKN linker sequence (SEQ ID NO:5) at the 3′ end resulting in an Xba1-L-C1-L-Xho1 DNA fragment. pRR21 has the following structure: S-C2N-L-Xho1-T1-L-Xba1-L-C1-L-Xho1—C2C-V. In addition this construct was inserted into pAM9 before additional insertions were made. The DNA sequence encoding GFP was PCR-amplified with Xba1 ends (no 3′ linker sequence) and inserted into the Xba1 site between T1 and C1 of pRR21 to produce MGEV-7. MGEV-7 was inserted into pBIN19 to produce the vector PHEX 46, diagrammed in
Transient Expression in Tobacco Leaves
pHEX 46 was introduced into A. tumefaciens and the expression of T1, C1 and GFP was determined by a transient assay with tobacco leaves. The method was that essentially described in Example 2 for cotton cotyledons except that Nicotiana benthamiana plants were grown for 5 weeks in a controlled temperature growth cabinet (25° C., 16 h/8 h light/dark cycle). The underside of leaves (4-6 nodes from the top, 6-10 cm in maximum width) was infiltrated by gently pressing a 1 mL syringe and filling the leaf cavity with the Agrobacterium suspension. Four to six infiltrations were made on each leaf. Plants were grown for a further 4 days. The infiltrated areas were then cut out, weighed and frozen in liquid nitrogen. Protein expression was determined by immunoblots as described in Example 1.
Transient Expression in Cotton Cotyledons
Expression of PHEX 46 was also determined in a transient assay with cotton cotyledons as previously described in Example 2.
Protein Detection
Expression of NaPI was determined by ELISA as described in Example 1.
Immunoblot analysis was carried out as described in Example 1 with the modification described in Example 2.
Microscopy
Three days after infiltration with A. tumefaciens the N. benthamiana and cotton plants were placed in the dark for 24 h. The infiltrated leaf areas were then removed and epidermal peels (˜5 mm2) were prepared. Small pieces (1-2 mm2 of the epidermal or mesodermal tissue were placed on a glass slide with water as a mounting medium. A cover slip was placed over the top and sealed with hot wax. The sections were examined for GFP fluorescence using an Olympus BX50 fluorescence microscope. A W1 B filter (excitation range 460-490 nm) was used for fluorescence excitation and a long pass filter which detects signals at 515 nm plus was used for emission. GFP fluorescence was also examined using a Leica TCS SP2 confocal laser-microscope. The Argon laser excitation wavelength was 488 nm; GFP emission was detected with the filter set for FITC (505-530 nm).
Results
Several transient assays with both tobacco leaves and cotton cotyledons were conducted. NaPI was detected by ELISA in cotton cotyledons (
Immunoblot analysis of tobacco leaf extracts after transient expression confirmed that the GFP protein was present (
GFP produced from transient expression of MGEV-7 in the epidermal cells of cotton leaves was located in the vacuole (
The results demonstrate that proteins of disparate sizes can be expressed as a polyprotein using a MGEV, and correctly processed after translation to yield individual protein components. In this example, 4 proteins ranging in size from ˜6 kDa to ˜28 kDa were effectively expressed together and correctly processed. A single vacuole targeting sequence resulted in transfer of each expressed protein to the cell vacuole prior to processing. The use of GFP in a MGEV is therefore a convenient means to indicate intracellular location of proteins co-expressed in a MGEV.
The MGEV described in this example (MGEV-9) has the structure diagrammed as:
(See
MGEV-9 expressing six proteins, a defensin, two potato type one PI's and 3 type two PI's was constructed using the following method. NaD1 was prepared as per Example 3. The Pot 1A dimer was constructed by splice overlap PCR. The first Pot 1A was PCR-amplified with a 5′ XbaI site and a 3′ linker sequence. The second Pot 1A was PCR-amplified with linker sequences at both ends and a 3′ XbaI site. The two PCR fragments were annealed to each other and extended for 8 cycles; outer primers were then added to PCR-amplify the dimer sequence. The NaD1 and Pot 1A dimer fragments were inserted into the Xba 1 site of pSP1 (Example 2) in a 3 way ligation. The new larger fragment (T1-NaD1-Pot 1A-Pot 1A-C1) was cut at the Xho 1 sites to produce MGEV-9. MGEV-9 was inserted into pBIN19 to produce the vector pHEX55, diagrammed in
Transient Expression in Cotton Cotyledons
Expression of pHEX55 was determined in a transient assay with cotton cotyledons as previously described in Example 2.
Protein Detection
Expression of NaPI was determined by ELISA as described in Examples 1, 2 and 3.
Immunoblot analysis was carried out as described in Example 1 with the modification described in Example 2.
Results
NaPI (
The results demonstrate simultaneous expression and correct processing of several different proteins in a 6-domain circular MGEV.
The MGEV described in this example has the structure diagrammed as:
(See
This MGEV (MGEV-10) was essentially the same as MGEV-7 (Example 4) except that it did not have the NaPI vacuole targeting peptide (V) and the multipurpose vector pRR20 was used (Example 3). pRR20 was PCR-amplified using a reverse primer which excluded the vacuole targeting peptide (V). XbaI-flanked GFP was then ligated into the XbaI site. Details of the GFP are given in Example 4. The fragment (S-C2N-T1-GFP-C1-C2C) was then inserted into pAM9 to produce MGEV-10. MGEV-10 was then inserted into pBIN19 to produce the vector pHEX45, diagrammed in
Transient Expression Assays
Expression of pHEX45 was determined in transient assays with tobacco leaves and cotton cotyledons as described in Example 4. Protein expression was determined by immunoblots as described in Example 4. Two non-MGEV constructs C1 and C2 (
Results
Several transient assays with tobacco leaves were conducted. Immunoblot analysis of tobacco leaf extracts after transient expression confirmed that the GFP protein was produced from both the control constructs (C1 and C2) and was the same size as the 28 kDa bacterially expressed GFP (
GFP produced from transient expression of MGEV-7 in the epidermal cells of cotton leaves was located in the vacuole (Example 4,
GFP was also directed extracellularly when MGEV-10 was expressed transiently in the leaves of N. benthamiana.
The results confirm and amplify those obtained in Example 4. Vacuolar targeting of GFP was observed regardless of whether the targeting sequence was directly attached to GFP protein or to the unprocessed MGEV protein.
The MGEV described in this example (MGEV-11) has the structure diagrammed as:
(See also
A MGEV expressing a defensin and 3 potato type two PI's was constructed, essentially as described for MGEV-7 (Example 4) except that NaD1 defensin included the C-terminal acidic peptide tail. NaD1CTPP, SEQ ID NO:14 amino acids 26-105 was inserted into the Xba1 site of the multipurpose vector pRR21 (Example 4) to produce MGEV-11. MGEV-11 was then inserted into pBIN19 to produce the vector PHEX 42, diagrammed in
Transient Expression in Cotton Cotyledons
Expression of PHEX 42 was determined in a transient assay with cotton cotyledons as described in Example 2.
Protein Detection
Protein expression was determined by ELISA and immunoblots as described in Example 4.
Results
NaPI (
The results demonstrate expression and correct processing of NaD1 having its own vacuolar targeting sequence (CTPP), in addition to the vacuole targeting sequence of the MGEV.
The MGEV described in this example has the structure diagrammed as:
(See
A MGEV expressing two potato type 1 PIs and 3 potato type two PI's was constructed, using pSP2 (Example 6). The Pot 1A dimer was produced as described in Example 5 and inserted into pSP2 to produce MGEV-12. MGEV-12 was then inserted into pBIN19 to produce the vector PHEX 33, diagrammed in
Transient Expression in Cotton Cotyledons
Expression of pHEX33 was determined in a transient assay with cotton cotyledons as described in Example 2.
Protein Detection
Protein expression was determined by ELISA.
Results
NaPI (
The results indicate that PotIA is expressed in a MGEV and correctly processed in concert with other proteins.
The MGEV described in this example has the structure diagrammed as:
(See
A MGEV expressing one class one defensin (NaD2) SEQ ID NO:15 and 16, one class two defensin (NaD1) SEQ ID NO:14, amino acids 26-72, and 3 type two PI's was constructed, essentially as described for MGEV-7 (Example 4) except that two defensins were inserted instead of GFP (see Lay, F. T., et al., (2005), Current Proteins and Peptide Science 6:85-101 for definition of one and class two defensins). NaD1 is described in Example 3. The NaD2-NaD1 dimer was constructed by splice overlap PCR. NaD2 was PCR-amplified with a 5′ XbaI site and a 3′ linker sequence. NaD1 was PCR-amplified with a linker sequence at the 5′ end and a 3′ XbaI site. The two PCR fragments were annealed to each other and extended for 8 cycles; outer primers were then added to PCR-amplify the dimer sequence. The NaD2-NaD1 dimer was inserted into pRR21 to produce MGEV-13. MGEV-13 was then inserted into pBIN19 to produce the vector pHEX39, diagrammed in
Transient Expression in Cotton Cotyledons
Expression of pHEX39 was determined in a transient assay with cotton cotyledons as described in Example 2.
Protein Detection
Protein expression was determined by ELISA as described in Example 3.
Results
NaPI (
The results demonstrate the value of using MGEV to express a plurality of plant protective proteins simultaneously.
The MGEV described in this example has the structure diagrammed as:
S-T1-Pot 1A-Pot1A-C1-V
(See
A linear MGEV expressing two potato type 1 PIs and 2 potato type two PI's was constructed, essentially as described for MGEV-8 (Example 2) except that two Pot 1As were inserted. The Pot 1A-Pot 1A dimer was produced by PCR overlap as described in Example 5 and inserted into the linear multipurpose vector pSP1 (Example 2) to produce MGEV-14. MGEV-14 was then inserted into pBIN19 to produce the vector PHEX 48, diagrammed in
Transient Expression in Cotton Cotyledons
Expression of pHEX48 was determined in a transient assay with cotton cotyledons as described in Example 2.
Protein Detection
Protein expression was determined by ELISA and immunoblots as described in Example 2.
Results
NaPI (
The results demonstrate that expression of a linear MGEV protein is at least as effective for expressing multiple proteins as the circular form. MGEV efficacy does not depend on the presence of a “clasp” protein.
The MGEV described in this example has the structure diagrammed as:
S-T1-NaD1-C1-V
(See
A linear MGEV expressing one defensin (NaD1) SEQ ID NO:14 amino acids 26-72 and 2 potato type two PI's (T1 and C1) was constructed, essentially as described for MGEV-8 (Example 2) except that a defensin (NaD1) was inserted instead of Pot 1A. NaD1 (described in Example 3) was inserted into the linear multipurpose vector pSP1 (Example 2) to produce MGEV-15. MGEV-15 was then inserted into pBIN19 to produce the vector PHEX 47, diagrammed in
Transient Expression in Cotton Cotyledons
Expression of pHEX47 was determined in a transient assay with cotton cotyledons as described in Example 2.
Protein Detection
Protein expression was determined by ELISA and immunoblots as described in Example 3.
Results
NaPI (
The results further demonstrate efficacy of simultaneously expressing multiple proteins having disparate functions using a linear MGEV lacking coding sequences for cyclization of the expressed poly-protein.
The MGEV described in this example has the structure diagrammed as
S-ProPot 1A-Pot 1A
(See
A linear MGEV expressing 2 potato type one PIs was constructed by splice overlap PCR. The first fragment consisting of the Pot 1A signal sequence, prodomain (SEQ ID NO:20) (Pro) and mature domain PotIA (SEQ ID NO:11, herein) was PCR amplified with a 5′ Bam H1 site and a 3′ linker sequence. The second fragment consisting of the mature Pot 1A was PCR amplified with a 5′ linker sequence and a stop codon (TAA) followed by a Sal 1 site at the 3′ end. The two PCR fragments were annealed to each other and extended for 8 cycles; outer primers were then added to PCR-amplify the complete sequence. The S-ProPot 1A-Pot 1A fragment was then inserted into pAM9 to produce MGEV-16. MGEV-16 then inserted into pBIN19 to produce the vector pHEX35, diagrammed in
Transient expression in cotton cotyledons
Expression of pHEX35 was determined in a transient assay with cotton cotyledons as described in Example 2.
Protein Detection
Expression of Pot 1A was determined by ELISA as described in Example 1 except that a different Pot 1A antibody was used. The antibody was produced using a bacterially expressed C1-PotIA dimer (the C1 domain is from NaPIii SEQ ID NO:1 aa 54 to 106) and can detect both the C1 and PotIA proteins. This antibody is better at detecting Pot 1A than the Pot 1A specific antibodies described in Examples 1 and 2, however the C1-Pot 1A antibody can only be used when Pot 1A protein is expressed without the presence of the NaPI peptides. The primary C1-Pot 1A antibody and the secondary C1-Pot 1A-biotin antibody were used at 100 ng/well.
An immunoblot to detect Pot 1A was carried out as described in Example 1 with the modification described in Example 2. The primary C1-Pot 1A antibody was diluted 1:2,000 dilution from a 1 mg/ml stock and the secondary antibody (goat anti-rabbit IgG conjugated to horseradish peroxidase) was used at a 1:50,000 dilution.
Results
Pot 1A was detected by ELISA in cotton cotyledons transfected with pHEX 35 (
Immunoblot analysis using the C1-Pot 1A antibody confirmed that the Pot 1A protein was present (
The results further corroborate expression of PotIA in a MGEV-like structure and show that the propeptide on Pot 1 which is a vacuolar targeting sequence is proteolytically removed.
The MGEV described in this example has the structure diagrammed as:
S-T1-NaD1CTPP
(See
A linear MGEV expressing one potato type two PI (T1) and one defensin (NaD1) with C-terminal tail (CTPP) was constructed. NaD1 CTPP (See example 7) was PCR amplified with a 5′ Xba 1 site and a 3′ Sal 1 site. This fragment was inserted into the Xba 1-Sal 1 cut site of pSP1 (with C1-V removed) to produce MGEV-17. MGEV-17 was then inserted into pBIN19 to produce the vector pHEX41, diagrammed in
Transient Expression in Cotton Cotyledons
Expression of pHEX41 was determined in a transient assay with cotton cotyledons as described in Example 2.
Protein Detection
Expression of NaD1 was determined by ELISA and immunoblots as described in Example 3.
Results
NaPI and NaD1 were detected by ELISA in cotton cotyledons transfected with pHEX41 (
Immunoblot analysis using the NaPI antibody confirmed that the processed NaPI 6 kDa peptides were present (
The MGEV described in this example has the structure diagrammed as:
S-NaD2-NaD1 CTPP
A linear MGEV expressing one class one defensin (NaD2) and one class two defensin (NaD1 with C-terminal tail) was constructed by splice overlap PCR essentially as described in Example 13 except that two defensins were used. NaD2 is described in Example 10 and NaD1-CTPPIs described in Example 7. The first fragment consisted of the signal sequence and the coding sequence for NaD2, the second fragment consisted of the mature NaD1 and the CTPP tail from NaD1. Following PCR, the full fragment (S-NaD2—NaD1 CTPP) was inserted into pAM9 to produce MGEV-18. MGEV-18 was then inserted into pBIN19 to produce the vector pHEX52, diagrammed in
Transient Expression in Cotton Cotyledons
Expression of pHEX52 was determined in a transient assay with cotton cotyledons as described in Example 2.
Protein Detection
Expression of NaD1 was determined by ELISA as described in Example 3.
Results
NaD1 was detected by ELISA in cotton cotyledons transfected with pHEX52 (
The MGEV described in this example has the structure diagrammed as:
S-NaD2-NaD1
A linear MGEV expressing one class one defensin (NaD2) and one class two defensin (NaD1) but lacking the CTPP tail was constructed as described in Example 15 except that the CTPP tail was not amplified. The S-NaD2-NaD1 fragment was inserted into pAM9 to produce MGEV-19. MGEV-19 was then inserted into pBIN19 to produce the vector pHEX51, diagrammed in
Transient Expression in Cotton Cotyledons
Expression of pHEX51 was determined in a transient assay with cotton cotyledons as described in Example 2.
Protein Detection
Expression of NaD1 was determined by ELISA as described in Example 3.
Results
NaD1 was detected by ELISA in cotton cotyledons transfected with pHEX51 (
The MGEV described in this example has the structure diagrammed as:
S-T1-GUSC1-V
A linear MGEV expressing one GUS and 2 potato type two PI's (T1 and C1) was constructed, essentially as described for MGEV-8 (Example 2) except that a DNA sequence encoding beta-Glucuronidase (GUS) was inserted in place of Pot 1A. GUS is an E. coli enzyme with a molecular mass of approximately 68,000 Da and is encoded by the gusA gene, SEQ ID NO:18 and SEQ ID NO:19 for GUS DNA and amino acid sequences, respectively. GUS was PCR amplified from the binary vector pBI121 (Invitrogen) with Xba 1 sites at each end, and inserted into the linear multipurpose vector pSP1 (Example 2) to produce MGEV-20. MGEV-20 was then inserted into pBIN19 to produce the vector pHEX58, diagrammed in
Transient Expression in Cotton Cotyledons
Expression of pHEX58 was determined in a transient assay with cotton cotyledons as described in Example 2.
Protein Detection
Expression of NaPI was determined by ELISA as described in Example 1
Immunoblot analysis to detect the NaPIs was carried out as described in Example 1 with the modification described in Example 2.
Results
NaPI was detected by ELISA in cotton cotyledons transfected with PHEX 58 (
Immunoblot analysis using the NaPI antibody confirmed that the mature NaPI peptides were present (
As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term “comprising”, particularly in a description of components of a composition or in a description of elements of a device, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or elements. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.
The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
In general the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art. The following definitions are provided to clarify their specific use in the context of the invention.
All patents and publications mentioned in the specification are incorporated by reference to the extent there is no inconsistency with the present disclosure, and those references reflect the level of skill of those skilled in the art to which the invention pertains.
One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent in the present invention. The methods, components, materials and dimensions described herein as currently representative of preferred embodiments are provided as examples and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention will occur to those skilled in the art, are included within the scope of the claims.
Although the description herein contains certain specific information and examples, these should not be construed as limiting the scope of the invention, but as merely providing illustrations of some of the embodiments of the invention. Thus, additional embodiments are within the scope of the invention and within the following claims.
| Number | Date | Country | |
|---|---|---|---|
| 60803206 | May 2006 | US |
