Patent Application
20120070900
The present disclosure relates to transformation of plant cells. In particular, the present disclosure relates to a DNA/protein nano-complex comprising an expressible nucleotide sequence for transforming plant cells. The present disclosure also pertains to methods for preparing the DNA/protein nano-complex, and to methods for transforming plant cells.
Transgenesis, also referred to as genetic transformation, allows for the generation of plants with improved traits significantly faster than any conventional breeding practice. This technology is based on the delivery of genes of interest from a broad range of sources into a plant genome. Two major transformation techniques include Agrobacterium-mediated DNA delivery and biolistic DNA transfer. Agrobacterium-mediated transformation relies on the ability of Agrobacterium tumefaciens to transfer a portion of its DNA, called transferred DNA or T-DNA, into plant cells. During its transit from the bacterial cell to the plant nucleus, the single-stranded T-DNA is protected by a single-stranded binding protein (VirE2) and guided by the VirD2 protein. The latter is important for bringing the T-DNA into the nucleus and possibly for integrating it into the genome. The integration process apparently requires broken DNA or at least an area of active replication or transcription. Agrobacterium-mediated transformation is an efficient process and typically in dicotyledonous (dicots) plants predominantly results in integration of the transgenes at single locus; integrated T-DNA is mostly intact and allows normal expression of the transgene. However, transformation of monocotyledonous (monocots) plants with Agrobacterium tumefaciens is not very efficient. This may be due to inability of Agrobacteria to efficiently attach to the cell wall of monocots. This creates substantial problems, since quite a number of important agricultural crops are monocots (wheat, corn, triticale, barley, rye etc.). In addition, most of the vectors used for Agrobacterium-mediated transformation have common vector backbones and thus are frequent targets of rearrangements occurring prior to integration. This creates complex transgene integration patterns.
An alternative method used for transforming monocots is based on simple gold-particle mediated bombardment of naked DNA into plant tissue. The DNA is not protected against endonucleases during such biolistic transformation and the technique relies on host import proteins to transfer the DNA inside the nucleus. Hence biolistic transformation is inefficient and may generate multiple integrations of truncated, duplicated and/or rearranged transgenes.
It has been suggested that a specifically designed DNA/protein complex may be used for transforming plant cells (e.g., WO 95/05471) or animal cells (e.g., U.S. Pat. No. 6,498,011). The complex contains a chimeric recombinant DNA construct covalently associated with a VirD2 protein. The complex can be accompanied by further Vir proteins such as VirE2. It has been suggested that VirE2 may aid in the transfer of the complex through the plant cell plasma membrane (2001, Dumas F. et al., An Agrobacterium VirE2 channel for transferred-DNA transport into plant cells. Proc. Natl. Acad. Sci. USA, 98: 485-490).
While a DNA/VirD2 complex might be able deliver a DNA molecule to the nucleus of a target cell, the complex is not protected from, for example, endonucleases in the cytoplasm. In Agrobacterium-mediated transformations, the DNA is protected by VirE2 and, hence, it has been suggested to add this protein to the complex. However, for a variety of reasons, VirE2 is difficult to purify in useful quantities.
The present disclosure provides novel T-DNA/protein nano-complexes useful for transforming plant cells. The T-DNA/protein nano-complexes can be prepared in vitro. The nano-complexes protect associated T-DNA molecules from degradation during and after delivery to target plant cells. The present disclosure further provides a method of transforming plant cells using the T-DNA/protein nano-complexes. The present approach may lead to integration of fewer and more intact copies of delivered DNA molecules. The present approach may be a suitable alternative to the bombardment techniques currently used for monocots.
This summary does not necessarily describe all features of the invention. Other aspects, features and advantages of the invention will be apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention.
a) is a micrograph of a gel showing the cleavage activity of the recombinant VirD2 protein in the presence or absence of 1 μg of VirD2, and
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In order that the invention herein described may be fully understood, the following terms and definitions are provided herein.
As used herein, the term “synthetic DNA” means DNA sequences that have been prepared entirely or at least partially by chemical means. Synthetic DNA sequences may be used, for example, for modifying native DNA sequences in terms of codon usage and expression efficiency.
The word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or group of integers.
As used herein, the word “complexed” means attached together by one or more linkages.
The term “a cell” includes a single cell as well as a plurality or population of cells.
The term “about” or “approximately” means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.
The term “nucleic acid” refers to a polymeric compound comprised of covalently linked subunits called nucleotides. Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded. DNA includes cDNA, genomic DNA, synthetic DNA, and semisynthetic DNA.
The term “gene” refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids.
The term “recombinant DNA molecule” refers to a DNA molecule that has undergone a molecular biological manipulation.
The term “vector” refers to any means for the transfer of a nucleic acid into a host cell. A vector may be a replicon to which another DNA segment may be attached so as to bring about the replication of the attached segment. A “replicon” is any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control. The term “vector” includes plasmids, DNA-protein complexes, and biopolymers. In addition to a nucleic acid, a vector may also contain one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).
The term “cloning vector” refers to a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. Cloning vectors may be capable of replication in one cell type, and expression in another (“shuttle vector”).
A cell has been “transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell. A cell has been “transformed” by exogenous or heterologous DNA when the transfected DNA effects a phenotypic change. The transforming DNA can be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
The term “nucleic acid molecule” refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.
Modification of a genetic and/or chemical nature is understood to mean any mutation, substitution, deletion, addition and/or modification of one or more residues. Such derivatives may be generated for various purposes, such as in particular that of enhancing its production levels, that of increasing and/or modifying its activity, or that of conferring new pharmacokinetic and/or biological properties on it. Among the derivatives resulting from an addition, there may be mentioned, for example, the chimeric nucleic acid sequences comprising an additional heterologous part linked to one end, for example of the hybrid construct type consisting of a cDNA with which one or more introns would be associated.
Likewise, for the purposes of the invention, the claimed nucleic acids may comprise promoter, activating or regulatory sequences, and the like.
The term “promoter sequence” refers to a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
A coding sequence is “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced (if the coding sequence contains introns) and translated into the protein encoded by the coding sequence.
The term “homologous” in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a “common evolutionary origin,” including homologous proteins from different species. Such proteins (and their encoding genes) have sequence homology, as reflected by their high degree of sequence similarity. This homology is greater than about 75%, greater than about 80%, greater than about 85%. In some cases the homology will be greater than about 90% to 95% or 98%.
“Amino acid sequence homology” is understood to include both amino acid sequence identity and similarity. Homologous sequences share identical and/or similar amino acid residues, where similar residues are conservative substitutions for, or “allowed point mutations” of, corresponding amino acid residues in an aligned reference sequence. Thus, a candidate polypeptide sequence that shares 70% amino acid homology with a reference sequence is one in which any 70% of the aligned residues are either identical to, or are conservative substitutions of, the corresponding residues in a reference sequence.
The term “polypeptide” refers to a polymeric compound comprised of covalently linked amino acid residues. Amino acids are classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group. A polypeptide of the invention preferably comprises at least about 14 amino acids.
The term “protein” refers to a polypeptide which plays a structural or functional role in a living cell.
The term “VirD2” refers to the VirD2 protein which is useful for integrating T-DNA into plant genomes as described by Ziemienowicz et al. (2008, Mechanisms of T-DNA integration. In: Tzfira et al. (Eds.) Agrobacterium: from biology to biotechnology. pp 396-441. Springer, New York, USA).
The term “VirE2” refers to the VirE2 protein which protects single-stranded T-DNA during transfer of the T-DNA from microbial cells to plant nuclei as described by Rossi et al. (1996, Integration of complete transferred DNA units is dependent on the activity of virulence E2 protein of Agrobacterium tumefaciens. Proc. Natl. Acad. Sci. USA, 93:126-130).
The term “corresponding to” is used herein to refer to similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured. A nucleic acid or amino acid sequence alignment may include spaces. Thus, the term “corresponding to” refers to the sequence similarity, and not the numbering of the amino acid residues or nucleotide bases.
The term “derivative” refers to a product comprising, for example, modifications at the level of the primary structure, such as deletions of one or more residues, substitutions of one or more residues, and/or modifications at the level of one or more residues. The number of residues affected by the modifications may be, for example, from 1, 2 or 3 to 10, 20, or 30 residues. The term derivative also comprises the molecules comprising additional internal or terminal parts, of a peptide nature or otherwise. They may be in particular active parts, markers, amino acids, such as methionine at position −1. The term derivative also comprises the molecules comprising modifications at the level of the tertiary structure (N-terminal end, and the like). The term derivative also comprises sequences homologous to the sequence considered, derived from other cellular sources, and in particular from cells of human origin, or from other organisms, and possessing activity of the same type or of substantially similar type. Such homologous sequences may be obtained by hybridization experiments. The hybridizations may be performed based on nucleic acid libraries, using, as probe, the native sequence or a fragment thereof, under conventional stringency conditions or preferably under high stringency conditions.
The embodiments of the present invention relate to novel T-DNA/protein nano-complexes useful for delivering selected DNA molecules to target cells for the purpose of transforming the cells, and to methods for preparing the novel T-DNA/protein nano-complexes. The selected DNA may be either of homologous or heterologous origin with respect to the plant material involved or it may be of synthetic origin or both. The DNA sequence can be constructed from genomic DNA, from cDNA, from synthetic DNA, or hybrids thereof.
The DNA may be single-stranded. VirD2 is able to cleave single-stranded DNA. For processing double-stranded DNA (e.g. plasmids) additional proteins may be necessary such as those exemplified by VirD1.
The DNA may comprise a recognition sequence for VirD2. For example, the DNA may comprise the so called right border (RB) sequence from Agrobacterium pTi plasmid as disclosed by Ziemienowicz et al. (2000, Plant enzymes but not Agrobacterium VirD2 mediate T-DNA ligation in vitro. Mol. Cell. Biol. 20: 6317-6322).
The DNA preferably comprises the RB sequence which is 24 nucleotides in length (although shorter oligonucleotides (e.g. 17 nt) can be processed by VirD2. DNAs carrying the RB sequence are named hereinafter as “T-DNA”.
The DNA may be of any suitable size. For example, the DNA may be 10-6500 bases long, or 15-5000 bases long, or 25-4500 bases long, or 100-2500 bases long. In theory, the DNA may be up to 200,000 bases long, such as the entire pTi plasmid of Agrobacterium (T-DNA in the pTi plasmid is 20,000 by long). However, while not wishing to be bound by theory, it is believed that the cleavage efficiency is reduced for longer molecules.
Suitable for use herein is virtually any DNA composition that may be delivered to plant cells to ultimately produce fertile transgenic plants. For example, regulatory elements such as plant promoters; a sequence that acts as a signal to terminate transcription and allow for the poly-adenylation of the resultant mRNA; or a specific leader sequence which may, for example, increase or maintain mRNA stability and prevent inappropriate initiation of translation. It may be desirable to introduce DNA for genes or gene families which encode a desired traits for agricultural crops such as, but not limited to, herbicide resistance or tolerance (e.g. glycophosphate-resistance genes); insect resistance or tolerance; disease resistance or tolerance (viral, bacterial, fungal, nematode); stress tolerance and/or resistance, as exemplified by resistance or tolerance to drought, heat, chilling, freezing, excessive moisture, salt stress; oxidative stress; increased yields; food content and makeup; physical appearance; male sterility; drydown; standability; prolificacy; starch properties; oil quantity and quality; and the like. One may desire to incorporate one or more genes conferring any such desirable trait or traits, such as, for example, a gene or genes encoding herbicide resistance.
DNA may be introduced for the purpose of expressing RNA transcripts that function to affect plant phenotype yet are not translated into protein. Two examples are antisense RNA and RNA with ribozyme activity. Both may serve possible functions in reducing or eliminating expression of native or introduced plant genes.
DNA may be introduced for other purposes. For example, DNA elements including those of transposable elements such as Ds, Ac, or Mu, may be inserted into a gene and cause mutations. These DNA elements may be inserted in order to inactivate (or activate) a gene and thereby “tag” a particular trait.
In certain embodiments, the present invention contemplates the transformation of a recipient cell with more than one advantageous transgene. Two or more transgenes can be supplied in separate vectors, or alternatively, in a single vector that incorporates two or more gene coding sequences.
The T-DNA/protein nano-complexes of the present invention comprise VirD2. This protein is known to have a nuclear targeting function and can deliver ssDNA to the nucleus. However, when VirD2 is used alone, the DNA is unprotected from endonuclease activity which reduces the likelihood of a successful transformation. Furthermore, it is believed that VirD2 cannot aid in penetration of the complex through plant cell walls.
Suitable homologs of VirD2 may be used such as those exemplified by TraI from E. coli (Pansegrau et al., 1993, Site-specific cleavage and joining of single-stranded DNA by VirD2 protein of Agrobacterium tumefasciens Ti plasmids: Analogy to bacterial conjugation. Proc. Natl. Acad. Sci. USA 90: 11538-11542). The VirD2 may be obtained by any suitable means. For example, the protein may be purified in accordance with the method taught by Ziemienowicz et al. (2001, Import of Agrobacterium T-DNA into plant nuclei: two distinct functions of VirD2 and VirE2 proteins. Plant Cell 13: 369-383).
The T-DNA/protein nano-complexes of the present invention comprise RecA, a protein isolated from Escherichia coli. RecA has a DNA-repair and maintenance function in E. coli. RecA is relatively easy to isolate and is available commercially (e.g., New England Biolabs, Ipswich, Mass., USA; Bio-Concept Laboratories Inc., Salem, N.H., USA). It has been suggested that RecA could serve as a substitute for the nuclear import function of VirE2 but that RecA cannot substitute for VirE2 in efficient T-DNA transfer (Ziemienowicz et al., 2001, Plant Cell 13: 369-383).
Surprisingly, despite the prior art teachings, it has been found that the T-DNA/protein nano-complexes of the present invention comprising RecA can efficiently transform plant cells.
Various homologs of RecA are known and may be used herein. For example, Tth RecA, yeast Rad51, or any other single-strand DNA-binding protein of prokaryotic or eukaryotic origin which forms filaments with similar structure as ssDNA-RecA filaments.
The present complex may be delivered to the target cells by any suitable means. Such techniques are known in the art and are exemplified by electroporation, bombardment, microinjection, liposomes, and the like.
The T-DNA/protein nano-complexes of the present invention may additionally comprise a compatible cell penetrating peptide exemplified by Tat2. Cell penetrating peptides (CPP) are a class of relatively short peptides that have the ability to translocate across cell membranes. Any suitable CPP may be used herein. For example, the T-DNA/protein nano-complexes may comprise a Tat2 peptide having an amino acid sequence RKKRRQRRRRKKRRQRRR (SEQ ID NO: 1). An alternative CPP system is described in U.S. Pat. No. 6,841,535. CPPs may be obtained from companies offering peptide synthesis service (e.g., Biomatik Corp., Cambridge, ON, Canada; Pacific Immunology Corpo, Ramona, Calif., USA; LifeTein LLC, South Plainfield, N.J., USA). Some CPPs are available in a form of kits (e.g., the Chariot Protein Delivery Kit from Active Motif, Carlsbad, Calif., USA).
While not wishing to be bound by theory, it is believed that the single stranded T-DNA molecule is covalently linked to VirD2. Then, the T-DNA/VirD2 complex is covered by RecA, a protein with high affinity for single stranded DNA. The ssT-DNA/VirD2/RecA complex is then linked to CPP in vitro, forming a nano-complex that has the ability to transfect plant cells. The CPP is believed to release the ssT-DNA/VirD2/RecA complex in the cytoplasm, and VirD2 guides the complex to the nucleus. The ssT-DNA is passively protected by RecA from nuclease activity in the cytoplasm. Upon reaching the nucleus, RecA protein dissociates and VirD2 assists the T-DNA in its integration into the genome.
The T-DNA/protein nano-complexes of the present invention may be assembled in any suitable manner known to those skilled in these arts. An exemplary process for assembling an exemplary T-DNA/protein nano-complex comprises the following steps:
The T-DNA/protein nano-complexes of the present invention may be used to transform any suitable plant cell target. For example, cells from angiosperms (dicots, monocots). The present complex may be particularly useful for transforming cells from monocots such as wheat, corn, triticale, barley, rye and the like.
The present T-DNA/protein nano-complexes may be delivered, for example, to spores derived from monocots, for example wheat microspores, corn microspores, triticale microspores, barley microspores, rye microspores, and the like. The greatest advantage of microspore regeneration is the ability to obtain double haploids and thus to faster obtain plants homozygous for the transgene than is possible with standard methods currently used by those skilled in these arts. Use of the T-DNA/protein nano-complexes of the present invention to transform target plant cells enables skipping an entire generation and the lengthy and costly routine of selection for the homozygous lines.
The transformation may be performed in any suitable manner known to those skilled in these arts. An exemplary process for transformation comprises the following steps:
Step 1: preparing a T-DNA/protein nano-complex according to the present disclosure; and
Step 2: exposing target plant cells to the T-DNA/protein nano-complex.
The present method may have a transformation efficiency of about 1% or greater, about 2% or greater, about 5% or greater, about 7% or greater, about 10% or greater, about 15% or greater, about 17% or greater, about 20% or greater, about 22% or greater, about 25% or greater. The efficiency of transformation may be calculated by dividing the number transgenic plants by the number of plants regenerated.
All citations are herein incorporated by reference, as if each individual publication was specifically and individually indicated to be incorporated by reference herein and as though it were fully set forth herein. Citation of references herein is not to be construed nor considered as an admission that such references are prior art to the present invention.
The invention includes all embodiments, modifications and variations substantially as hereinbefore described and with reference to the examples and figures. It will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims. Examples of such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way.
To form an exemplary T-DNA/protein nano-complex in vitro, a GUS expression cassette consisting of rice Actin promoter and intron, uidA (GUS) gene and nos terminator was amplified by PCR using primers annealing 200 by upstream (forward primer) and downstream (reverse primer) in a pACT-1D plasmid. The primers were designed to contain the Agrobacterium right border sequence (RB) in direct and inverted orientation at the 3′ and 5′ end of the GUS cassette, respectively (
Agrobacterium VirD2 protein was purified following the methods taught by Ziemienowicz et al. (2001). The recombinant VirD2 protein was produced in E. coli as 6×His fusion, and purified using affinity and ion-exchange chromatography following the method disclosed by Pelczar et al. (2004, Agrobacterium proteins VirD2 and VirE2 mediate precise integration of synthetic T-DNA complexes in mammalian cells. EMBO Rep. 5: 632-637).
ssT-DNA containing the Act_GUS_nos cassette was prepared generally following the methods taught by Ziemienowicz et al. (1999,) and Ziemienowicz et al. (2001). The DNA insert was produced by PCR using primers p1 and p2 containing RB sequence and NcoI site (Table 1) and pACT-1D plasmid as a template.
The PCR reaction mixture (25 μL) contained GC buffer, 0.2 mM dNTPs, 0.5 μM of each primer, 5 ng of DNA template and 0.5 U of high fidelity Phusion® DNA polymerase (Phusion is a registered trademark of Finnzymes Oy, Vantaa, Finland; the Phusion® DNA polymerase product was obtained from Fermentas Canada Inc., Burlington, ON, Canada). The amplification reactions consisted of a preliminary denaturation step at 98° C. for 30 s, followed by 10 cycles of 98° C. for 10 s, 59° C. for 30 s, 72° C. for 75 s, 20 cycles of 98° C. for 10 s, 67° C. for 30 s, 72° C. for 75 s followed by incubation at 72° C. for 10 min. The PCR product was extracted from agarose gel, purified using a QiaQuick® gel extraction kit (QuiQuick is a registered trademark of Qiagen GMBH, Hilden, Fed. Rep. Germany; the product was purchased from Qiagen Inc., Toronto, ON, Canada) and cloned into the NcoI site of LITMUS29 plasmid vector resulting in LITMUS29_iRB_PAct-GUS-Tnot
The VirD2-T-DNA complex was prepared as follows. The purified VirD2 protein was first tested for its cleavage activity using model oligonucleotides containing the RB sequence, following the method taught by Ziemienowicz et al. (2000, Mol Cell Biol. 20: 6317-6322). Optimization of the reaction was also performed by testing different salts and their concentration as well as various protein-to-oligonucleotide ratios. Under the most optimal conditions, the efficiency of the cleavage reaction was nearly 90% when 5-10 μg of VirD2 was used per 1 pmol of oligonucleotide. The VirD2-ssT-DNA complex was formed by reacting 2.0 μg of ssT-DNA with 10 μg of the VirD2 protein in TKM buffer (50 mM Tris-Cl pH 8.0, 150 mM KCl, 1 mM MgCl2) for 1 h at 37° C. Cleavage efficiency of VirD2 on ssT-DNA was ˜75%, and ssDNA binding efficiency of RecA was 100% (
Next, oligonucleotides used as primers for production of T-DNA (4.8 kb long NcoI/RB Act_GUS_nos_RB/NcoI cassette) by PCR were tested as the substrates for VirD2. The efficiency of the cleavage reaction was slightly lower than in the case of model oligonucleotide substrates, but still very high: 70-80% at the same protein:oligonucleotide ratio. Then, ssT-DNA (4.8 kb long RB Act_GUS_nos_RB cassette) was used as the substrate for VirD2. Cleavage efficiency of 75% was achieved by using 250 ng of VirD2 for 100 ng of DNA. The VirD2-T-DNA complex was then formed by reacting 2.0 μg of ssT-DNA with 10 μg of the VirD2 protein in TKM buffer (50 mM Tris-Cl pH 8.0, 150 mM KCl, 1 mM MgCl2) for 1 h at 37° C.
The VirD2-T-DNA-RecA complex was prepared as follows and illustrated in
The VirD2-T-DNA-RecA complex was then treated with CPP Tat2 following the method taught by Chugh et al. (2008, Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos. FEBS J. 275(10): 2403-2414). Tat2 peptide was added to the formed VirD2-T-DNA-RecA complex at the ratio of 4:1 (4 μg of peptide per 1 μg of DNA). The two components were mixed and incubated for 15 minutes at room temperature. Next, 5 μg of lipofectamine were added followed by incubation for 5 minutes at the same conditions, and the reaction efficiency monitored following the method taught by Ziemienowicz et al. (1999, Proc. Natl. Acad. Sci. USA 96: 3729-3733).
Triticale var. Ultima microspores were transfected with DNA or T-DNA/protein complexes in the presence or absence of the CPP Tat2 peptide (SEQ ID NO: 1) following the steps outlined in
Transfected microspores were cultured in 30-mm Petri dishes containing liquid NPB-99 medium supplemented with 10% Ficoll in the presence of 4 ovaries per plate. Plates were incubated for 4-6 weeks at 28.5° C. in the dark. Formed embryos that were 1-2 mm long (
Efficiency of regeneration of green plantlets varied between various samples from different treatments in all of the independent experiments as indicated by high standard deviation values. The average regeneration efficiency values did not exceed 10% in most cases, with the lowest value observed for transfection was with the full T-DNA/protein complex (VirD2-ssT-DNA-RecA) among all treatments in the presence of the Tat2 CPP (Table 1). In total, 303 plants were regenerated in vitro and transplanted into soil-less growing mix. The survival rate of the transplanted plantlets was 93%, which resulted in 281 plants successfully cultured in the soil-less growing mix (Table 2).
The presence of the GUS transgene in genomes of plants regenerated after transfection was determined by PCR analysis and confirmed by Southern blot analysis. Genomic DNA was isolated from 100 mg leaf samples using the cetyltrimethylammonium bromide (CTAB) method disclosed by Doyle et al. (1987, Preservation of plant samples for DNA restriction endonuclease analysis. Taxon 36: 715-772), modified according to the DArT protocol (http://www.diversityarrays.com/sites/default/files/pub/DArT_DNA_isolation.pdf). To detect the GUS gene using PCR methodology, GUS specific primers p3 and p4 (SEQ ID NO: 4 and SEQ ID NO: 5 respectively) were combined with Actin intron p5 specific primer (SEQ ID NO: 6) and nos terminator p6 specific primer (SEQ ID NO: 7) (Table 1;
PCR analysis revealed that omission of the Tat2 peptide from the nano-complex resulted in low transfection efficiency evidenced by no or very few GUS-positive plants (Table 3). In contrast, DNA and DNA-protein complexes were transfected efficiently into triticale microspores via Tat2 peptide (Table 3). The percentage of GUS-positive plants was comparable when complete T-DNA complex (VirD2-ssT-DNA-RecA), ssT-DNA-VirD2 or linear naked dsDNA (pACT-1D/PstI, dsTDNA) were used for transfection (Table 2). Slightly lower values were observed for the ssT-DNA-RecA complex, whereas use of naked ssT-DNA generated low number of GUS-positive plants (Table 3), most likely due to the lack or incomplete protection of DNA from nucleases. Among naked DNA molecules, dsDNA was protected from nucleolytic degradation better than ssDNA (Table 3). All the control untreated plants were GUS-negative and only very few GUS-positive plants were found among those treated without DNA (Table 3). The latter ones likely represent false positive cases.
Next, Southern blot analysis was performed to verify PCR results. gDNA from GUS-positive triticale lines was digested with BamHI and XbaI and probed first with the GUS-specific probe and then with a probe specific for wheat EF1α gene. Transgenic and non-transformed triticale genomic DNA was isolated as described above and treated with RNaseA (final concentration: 80 μg/mL) for 10 min at 65° C., followed by purification using the phenol-chloroform method and precipitation with ethanol. gDNA was then digested using restriction enzymes: (a) BamHI and XbaI to test for the transgene presence, and (b) BamHI alone to test for the transgene copy number in a 500-μL reaction mixture containing NEB#3 buffer, 1 mg/mL BSA, 30 μg of gDNA and 400 U of the restrictase. The reactions were incubated at 37° C. over night. Digested DNA was purified using the phenol-chloroform method and concentrated by precipitation with ethanol. Southern blot analysis was performed following a modification to the protocol taught by Sambrook et al. (2001, Molecular Cloning: a Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y., USA). Twenty μg of digested gDNA were separated on a 0.8% agarose gel at 35V for 16 h in 1×TAE buffer. The gel was rinsed in dH2O, de-purinated for 15 min in 0.25N HCl, rinsed in dH2O, denatured for 30 min in 0.4N NaOH, rinsed again in dH2O, neutralized for 15 min in 0.5 M Tris-HCl pH 7.5 containing 3 M NaCl and soaked in transfer buffer (10×SSC: 1.5 M NaCl, 0.15 M sodium citrate) for 10 min. DNA transfer onto a positively charged nylon membrane was performed for 2.5 h using a vacuum blotter. DNA was then cross-linked to the positively charged nylon membrane at 120 mJ/cm2 in Spectrolinker Crosslinker (Spectromics Corp., Westbury, N.Y., USA). Probes were prepared using a PCR DIG Probe Synthesis kit (Roche Diagnostics, Laval, QC, Canada) and following to the protocol provided by the supplier. p18 and p6 primers were used for the GUS-specific probe and p19 and p20 primers were used for the EF1α-specific probe. Hybridization was carried out using DIG Easy Hyb solution (Roche Diagnostics) at 42° C. (GUS probe) or 65° C. (EF1α probe). Detection was performed using AP-conjugated anti-DIG antibodies (Roche Diagnostics) diluted 1:2,500 in blocking solution containing 1% Blocking Reagent (Roche Diagnostics) in maleic acid buffer (0.1 M maleic acid, 0.5 M NaCl, pH 7.5) and CPD Star (Roche Diagnostics) as a substrate. Images of the membrane were taken with Fluor Chem HD2 (Convergent Bioscience, Toronto, ON, Canada).
Presence of the GUS gene was confirmed in all transformed lines but not in Ultima wild type plants and plant lines regenerated from microspores transfected without DNA (e.g., line #41;
Next, GUS-positive plants were analyzed for the intactness of the integrated T-DNA. Analysis of the intactness of the integrated transgene cassette was performed by PCR using ten sets of primers specific to various regions of the GUS cassette (
Analysis of transgene expression was performed at the protein level using Western blotting technique on crude extracts from GUS-positive plants. GUS transgene was expected to be expressed in lines #23, 61, 63, 152 and 190 (from treatment with dsT-DNA+Tat2), 55 (ssT-DNA+Tat2 treatment), 51, 88 and 272 (ssT-DNA-RecA+Tat2 treatment), 137, 143, 254 and 265 (VirD2-T-DNA+Tat2 treatment) as well as 225, 237, 241, 267 and 269 (VirD2-T-DNA-RecA+Tat2 treatment). Crude protein extracts were prepared from leaf tissue (100 mg) following the method taught by Stoger et al. (1999, Expression of the insecticidal lectin from snowdrop (Galanthus nivalis agglutinin; GNA) in transgenic wheat plants: effect on predation by the grain aphid Sitobion avenae. Mol. Breed. 5: 65-73) using extraction buffer supplemented with Complete Protease Inhibitor Cocktail (Roche Diagnostics). Aliquots of 10 μg of total protein were analyzed by Western blotting in following a standard protocol disclosed by Sambrook et al. (2001) using polyclonal rabbit antibodies raised against the N-terminal peptide of bacterial β-glucuronidase (1:2,000; primary antibody; Abcam, Cambridge, Mass., USA) and donkey antibody to rabbit IgG (HRP conjugate; 1:10,000; secondary antibody; Abcam). Detection was carried out with ECL Plus Western blotting detection reagents according to the manufacturer's recommendations (GE Healthcare Biosciences, Uppsala, Sweden). Membranes were then exposed to X-ray films and the intensity of the signals was quantified using ImageJ software.
Results of the Western blot analysis showed that the bacterial β-glucuronidase protein was detected in all lines predicted to express the transgene according to PCR and Southern blot analyses (
It is known that standard procedures of plant transformation using Agrobacterium often result in clear integration patterns exemplified by low number of copies of integrated transgenes (Windels et al., 2008, Agrobacterium tumefaciens-mediated transformation: patterns of T-DNA integration into the host genome. In: Tzfira et al. (Eds.) Agrobacterium: from biology to biotechnology pp 442-483), whereas most other methods used in plant biotechnology such as direct gene transfer, bombardment, and the like, result in integration of multiple DNA molecules and, as consequence, multiple copy/multiple loci insertion patters, that may lead to variations in the transgene expression (Latham et al., 2006, The mutational consequences of plant transformation. J. Biomed. Biotech. 25376: 1-7). The transgene copy number and integration pattern were analyzed by Southern blotting using gDNA of transgenic plants expressing the transgene at the detectable level. gDNA was digested with BamHI and hybridized with the GUS-specific probe. Application of the CPP-mediated transgene delivery resulted in low copy number (≦5) and relatively simple patterns of transgene integration (
Finally, the transgene copy number and expression level were compared. The comparison revealed, in most cases, a clear correlation between these two factors (Table 4). Two lines containing a single copy of the transgene (line #267 and 88) showed very low protein levels. In the instance of another line (#272), low level of GUS expression is most likely caused by gene silencing induced by additional copies of the transgene. Variations in the transgene protein level were noted also in plants regenerated from microspores transfected with the complete nano-complex (VirD2-ssT-DNA-RecA+Tat2;
In summary, we developed novel T-DNA/protein nano-complexes and novel methods for the uses thereof for plant transformation. Furthermore, we have shown that these T-DNA/protein nano-complexes and related methods of use are suitable for plant species that are difficult to transform with the classical Agrobacterium-mediated techniques. Moreover, CPP-mediated delivery of the T-DNA complex results in Agrobacterium-like type of transgene integration pattern regarding transgene intactness, copy numbers generated, and expression efficiency. Moreover, our T-DNA/protein nano-complex strategy yields more frequent integration of intact transgene molecules into a single locus of a monocot genome resulting in efficient expression of the transgene in transgenic plants.
| Number | Date | Country | |
|---|---|---|---|
| 61350774 | Jun 2010 | US |
