Patent Application
20140101792
Over the expanse of time since the endosymbiotic events that led to the establishment of plant organelles, plant cells have evolved elaborate mechanisms to coordinate the expression of plastid genes with the changing developmental and functional requirements of the cell. In addition to the nuclear encoded, plastid localized RNA polymerase (NEP), the nucleus controls the expression of a suite of σ-factors required for the active transcription of photosynthetic genes by the plastid encoded RNA polymerase (PEP), with PEP itself being transcribed by NEP (Allison et al., 1996; Hess and Borner, 1999). Nuclear control over translation of plastid mRNA is exerted through the activities of numerous plastid-localized RNA binding proteins (RBPs). RBPs appear to have a tight affinity for their cognate sequences in plastid mRNAs and studies have demonstrated that their interactions are specific for particular genes (Nakamura et al., 1999, 2001; Shen et al., 2001; Meierhoff et al., 2003; Schmitz-Linneweber et al., 2005, 2006).
Plastid mRNAs, expressed as mono- or polycistrons, contain 5′ and 3′ untranslated regions (UTRs). Detailed analyses have demonstrated that within these UTRs lie cis elements, often forming secondary structures, which facilitate the interaction with nuclear encoded RBPs (Yang et al., 1995; Hirose and Sugiura, 1996; Klaff et al., 1997; Alexander et al., 1998; Zou et al., 2003; Merhige et al., 2005;). RBPs display an array of functions including processing of polycistronic transcription units, RNA maturation and editing, transcript stability and turnover and the recruitment of additional protein factors involved in initiation of translation in response to the demands of the cell (Nickelsen, 2003; Schimtz-Linneweber and Barkan 2007). In contrast to the high level of conservation found within protein coding regions and ribosomal RNAs, intergenic and untranslated regions are highly variable in chloroplast genomes (Daniell et al., 2006; Saski et al., 2007; Timme et al., 2007)
Chloroplast transformation strategies have utilized both endogenous and foreign regulatory elements to facilitate high levels of foreign gene expression. Hybrid systems comprising a modified tobacco (Nicotiana tabacum) ribosomal operon promoter (Prrn) in conjunction with a translational control region (TCR) derived from the N. tabacum plastid-encoded rbcL gene or from bacteriophage T7 gene 10 (g10) to express foreign genes have been utilized in numerous species (Guda et al., 2000; Kuroda and Maliga, 2001; Ruhlman et al., 2007). Another approach incorporates the native psbA 5′ and 3′ UTRs into transformation constructs (Verma et al., 2008). The potential of psbA 5′ UTR stems from its important role in plastids. Photosystem II core protein D1 is a polytopic thylakoid membrane constituent with five membrane-spanning helices encoded by the plastid psbA gene (Marder et al., 1987). Expression of D1 is predominantly regulated at the level of translation and requires the participation of RBPs imported into plastids post-translationally from the cytoplasm. Photosystem II is highly susceptible to excessive light and the primary target of the damage is D1. If the core protein is not efficiently removed and replaced the result is impairment of electron transport, known as photoinhibition (Yamamoto, 2001). It is this cycle of turn over that makes the psbA 5′ UTR an attractive tool to enhance the level of foreign gene expression in transplastomic lines. The use of endogenous psbA regulatory elements has facilitated the generation of transplastomic N. tabacum lines with enhanced expression of a large number of soluble (Verma et al., 2008) and membrane proteins (Singh et al., 2009), generating transplastomic lines conferring desired agronomic traits (Bock, R., 2007; Daniell et al., 2005; Verma and Daniell, 2007) or expressing biopharmaceutical proteins and vaccine antigens (Davoodi-Semiromi et al., 2009).
However, overwhelming majority of foreign proteins has been expressed in N. tabacum chloroplasts. In order to advance this field, chloroplast genomes of several crop species should be transformed. In addition, rapid and reproducible oral delivery systems expressing vaccine antigens, autoantigens or biopharmaceuticals should be developed. There are two major limitations in accomplishing these goals.
Development of direct organogenesis system is important for establishment of efficient and reproducible plastid transformation systems in crops regenerated via organogenesis. In addition, improvement in our understanding of the role of endogenous or heterologous regulatory sequences in transgene expression in plastids is essential.
Upper frame, autoradiographs; lower frame; ethidium bromide stained rRNA. Lanes 1) Ls-g10-CP; 2) Nt-Nt-CP; 3) Ls-Nt-CP; 4) Nt-Nt-PA; 5) Nt-Ls-PA; 6) Ls-Ls-PA; 7) Ls-g10-PA. B) Desitometric quantitification for abundance of foreign transcripts in lines with heterologous regulatory elements relative to those with native elements.
According to one embodiment, the invention pertains to a stable plastid transformation and expression vector which comprises an expression cassette optimized for expression in a target plant species. The expression cassette includes a regulatory sequence that is endogenous to the target plant species, a heterologous polynucleotide sequence of interest, transcription termination sequence functional in said plastid, and flanking each side of the expression cassette, flanking DNA sequences which are homologous to a DNA sequence of the target plastid genome. The vector enables the stable integration of the heterologous coding sequence into the plastid genome of the target plant that is facilitated through homologous recombination of the flanking sequence with the homologous sequences in the target plastid genome. Optionally, the expression cassette optionally includes a heterologous marker sequence.
Certain embodiments disclosed herein are based on the inventor's development of direct plastid transformation and plant regeneration system that enables more efficient and reproducible plastid transformation, as well as the inventor's research of the role of endogenous or heterologous regulatory sequences in transgene expression in plastids is essential. Nucleotide variability in the regions upstream of chloroplast genes that comprise promoters and UTRs was investigated. Coding and non-coding sequences across 20 crop species representing most major clades of angiosperms including 4 grasses and 3 legumes were studied. Furthermore, L. sativa and N. tabacum transplastomic lines regulating transgenes with endogenous or heterologous regulatory elements were used to investigate RNA-protein interaction, foreign transcript accumulation and polyribosome association (polysome assay), and foreign protein accumulation. The inventor has discovered that species-specific optimization of plastid transformation vectors and optimization of growth hormone requirement has a significant impact on foreign gene expression and transformation efficiency.
In a specific embodiment, the invention pertains to a chloroplast expression vector that implements a regulatory sequence that is endogenous to the target plant species, where the regulatory sequence relates to a 5′ UTR sequence of a chloroplast gene. Unless specified otherwise, the term 5′ UTR is intended to pertain to a native 5′ UTR and is construed to pertain to variants or fragments thereof. In an even more specific embodiment, the regulatory sequence relates to a 5′ UTR sequence of the psbA chloroplast gene, or fragment or variant thereof.
Provided herein are accession numbers for sequenced plastid genomes of several different species, see Table 3. Also, Attachment A includes a listing of psbA genes for different target species and their location in the plastid genome, as well as the accession no. for the genome. The location of the psbA gene in the genome is noted in Attachment A for each entry. Moreover, using Arabidopsis thaliana as a specific example, as shown on page 1 of Attachment A, it shows the genome accession no. NC—000932.1 and the location of the gene at by 383..1444. Unless specified otherwise, the 5′ UTR sequence comprises 200 base pairs (bp) or less upstream of the translation initiation codon. This guideline of identifying the 5′ UTR sequence for the psbA genes can be applied to other chloroplast genes. Table 2 lists common genes encoded by chloroplast genomes. Thus, broadly speaking, the 5′ UTR of these noted genes may be used as regulatory sequences.
As noted above, when considering the psbA gene or other chloroplast genes, the 5′ UTR comprises sequences 200 by or less upstream of the translation initiation codon. In more specific embodiments, the 5′ UTR is 10-100 by upstream of the initiation codon. In an even more specific embodiment, the 5′UTR is 10-70 by upstream of the initiation codon.
According to certain embodiments, the target plant species include, but are not limited to, cereals such as barley, corn, oat, rice, and wheat, melons such as cucumber, muskmelon, and watermelon; legumes such as bean, cowpea, pea, peanut; oil crops such as canola and soybean; solanaceous plants such as tobacco, tuber crops such as potato and sweet potato, and vegetables like tomato, pepper and radish; fruits such as pear, grape, peach, plum, banana, apple, and strawberry; fiber crops like the Gossypium genus such as cotton, flax and hemp; and other plants such as beet, cotton, coffee, radish, commercial flowering plants, such as carnation and roses; grasses, such as sugar cane or turfgrass; evergreen trees such as fir, spruce, and pine, and deciduous trees, such as maple and oak. Of greatest present interest are the major economically important crops like maize, rice, soybean, wheat and cotton, and also including Lactuca sativa, or any of the crops provided on the attached tables whose genomes have been sequenced.
A “fragment” of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides that is capable of specific hybridization to a target of interest, e.g., a sequence that is at least 15 nucleotides in length. The fragments of the invention comprise 15 nucleotides, preferably at least 20 nucleotides, more preferably at least 30 nucleotides, more preferably at least 50 nucleotides, more preferably at least 50 nucleotides and most preferably at least 60 nucleotides of contiguous nucleotides of a polynucleotide of the invention. A fragment of a polynucleotide sequence can be used in antisense, gene silencing, triple helix or ribozyme technology, or as a primer, a probe, included in a microarray, or used in polynucleotide-based selection methods of the invention.
The polynucleotide fragments of the invention may be produced by techniques well-known in the art such as restriction endonuclease digestion and oligonucleotide synthesis.
A partial polynucleotide sequence may be used, in methods well-known in the art to identify the corresponding full length polynucleotide sequence. Such methods include PCR-based methods, 5′RACE (Frohman M A, 1993, Methods Enzymol. 218: 340-56) and hybridization-based method, computer/database-based methods. Further, by way of example, inverse PCR permits acquisition of unknown sequences, flanking the polynucleotide sequences disclosed herein, starting with primers based on a known region (Triglia et al., 1998, Nucleic Acids Res 16, 8186, incorporated herein by reference). The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template. Divergent primers are designed from the known region. In order to physically assemble full-length clones, standard molecular biology approaches can be utilized (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987).
Variant polynucleotide sequences preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity to a specified polynucleotide sequence. Identity is found over a comparison window of at least 20 nucleotide positions, preferably at least 50 nucleotide positions, more preferably at least 100 nucleotide positions, and most preferably over the entire length of the specified polynucleotide sequence. In the context of UTR sequences, the comparison window will naturally be upstream of the initiation codon of a given chloroplast gene.
Polynucleotide sequence identity can be determined in the following manner. The subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.5 [November 2002]) in bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250), which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.
The identity of polynucleotide sequences may be examined using the following unix command line parameters:
The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. The bl2seq program reports sequence identity as both the number and percentage of identical nucleotides in a line “Identities=”.
Polynucleotide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs (e.g. Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). A full implementation of the Needleman-Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice, P. Longden, I. and Bleasby, A. EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6. pp. 276-277) which can be obtained from http://www.hgmp.mrc.ac.uk/Software/EMBOSS/. The European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences on line at http:/www.ebi.ac.uk/emboss/align/.
Alternatively the GAP program may be used which computes an optimal global alignment of two sequences without penalizing terminal gaps. GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.
Polynucleotide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [November 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
The similarity of polynucleotide sequences may be examined using the following unix command line parameters:
The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an “E value” which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. The size of this database is set by default in the bl2seq program. For small E values, much less than one, the E value is approximately the probability of such a random match.
Variant polynucleotide sequences preferably exhibit an E value of less than 1×10−10 more preferably less than 1×10−20, more preferably less than 1×10−30, more preferably less than 1×10−40, more preferably less than 1×10−50 , more preferably less than 1×10−60, more preferably less than 1×10−70, more preferably less than 1×10−80, more preferably less than 1×10−90 , more preferably less than 1×10−100, more preferably less than 1×10−110, and most preferably less than 1×10−120 when compared with any one of the specifically identified sequences.
Variants and homologs of the nucleic acid sequences described above also are useful nucleic acid sequences. Alternatively, homologous polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions: 2×SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2×SSC, 0.1% SDS, 50° C. once, 30 minutes; then 2×SSC, room temperature twice, 10 minutes each homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.
Species homologs of polynucleotides referred to herein also can be identified by making suitable probes or primers and screening cDNA expression libraries. It is well known that the Tm of a double-stranded DNA decreases by 1-1.5° C. with every 1% decrease in homology (Bonner et al., J. Mol. Biol. 81, 123 (1973). Nucleotide sequences which hybridize to polynucleotides of interest, or their complements following stringent hybridization and/or wash conditions also are also useful polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed., 1989, at pages 9.50-9.51.
Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20° C. below the calculated Tm of the hybrid under study. The Tm of a hybrid between a polynucleotide of interest or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):
T
m=81.5° C.-16.6(log10 [Na+ ])+0.41(% G+C)−0.63(% formamide)−600/l),
where l=the length of the hybrid in basepairs.
Stringent wash conditions include, for example, 4×SSC at 65° C., or 50% formamide, 4×SSC at 42° C., or 0.5×SSC, 0.1% SDS at 65° C. Highly stringent wash conditions include, for example, 0.2×SSC at 65° C.
Relevant articles on genetic sequences is provided: proinsulin (Brousseau et al., Gene, 1982 March;17(3):279-89; Narrang et al, Can J Biochem Cell Biol. 1984 April;62(4):209-16; and Georges et al, Gene 27 (2), 201-211 (1984); and CTB (Shi et al, Sheng Wu Hua Hsueh Tsa Chih 9 (No.4), 395-399 (1993).
As used herein, the term “derivative” in the context of proteinaceous agent (e.g., proteins, polypeptides, peptides, and antibodies) refers to a proteinaceous agent that comprises an amino acid sequence which has been altered by the introduction of amino acid residue substitutions, deletions, and/or additions. The term “derivative” as used herein also refers to a proteinaceous agent which has been modified, i.e., by the covalent attachment of any type of molecule to the proteinaceous agent. For example, but not by way of limitation, an antibody may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivative of a proteinaceous agent may be produced by chemical modifications using techniques known to those of skill in the art, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Further, a derivative of a proteinaceous agent may contain one or more non-classical amino acids. A derivative of a proteinaceous agent possesses a similar or identical function as the proteinaceous agent from which it was derived. The term “derivative” in the context of a proteinaceous agent also refers to a proteinaceous agent that possesses a similar or identical function as a second proteinaceous agent (i.e., the proteinaceous agent from which the derivative was derived) but does not necessarily comprise a similar or identical amino acid sequence of the second proteinaceous agent, or possess a similar or identical structure of the second proteinaceous agent. A proteinaceous agent that has a similar amino acid sequence refers to a second proteinaceous agent that satisfies at least one of the following: (a) a proteinaceous agent having an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence of a second proteinaceous agent; (b) a proteinaceous agent encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding a second proteinaceous agent of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, or at least 150 contiguous amino acid residues; and (c) a proteinaceous agent encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the nucleotide sequence encoding a second proteinaceous agent. A proteinaceous agent with similar structure to a second proteinaceous agent refers to a proteinaceous agent that has a similar secondary, tertiary or quaternary structure to the second proteinaceous agent. The structure of a proteinaceous agent can be determined by methods known to those skilled in the art, including but not limited to, peptide sequencing, X-ray crystallography, nuclear magnetic resonance, circular dichroism, and crystallographic electron microscopy. In a specific embodiment, a derivative is a functionally active derivative.
Direct shoots emerged from the leaf explants 21 days after culture in optimized regeneration medium. The shoot regeneration response was variable with different combinations of auxin and cytokinin (Table 1). We observed that auxin or cytokinin alone did not initiate shoot regeneration. A combination of auxin (NAA) and cytokinin (BAP) was necessary to induce shoot regeneration in different cultivars. Maximal direct shoot initiation was observed from the leaf explants cultured in medium supplemented with 0.1 μg mL−1 NAA and 0.2 ug ml−1 BAP. Other combinations of PGRs induced callus formation and decreased shoot regeneration in all cultivars. We also observed differences in responsiveness among cultivars. Simpson Elite (LS) showed the greatest regeneration potential (average 4.3 shoots per explant), whereas Lentissima (LL) had the least response (0.43 shoots per explants). Regardless of the cultivar studied, the medium supplemented with 0.1 μg ml−1 NAA and 0.2 μg ml−1 BAP induced the greatest number of shoots (Table 1). Following LS in regeneration efficiency were Romaine (LR; 2.5), Great Lake (LG; 1.5), and Evola (LE; 1.2). Therefore, LS cultured on LR medium with 0.1 μg ml−1 NAA and 0.2 μg ml−1 BAP was chosen for all of our transformation experiments.
Sequences of IGS upstream from genes representing different functional groups were extracted from 20 complete plastid genomes representing most major clades of angiosperms, including 4 grasses and 3 legumes. Alignments were anchored by the inclusion of 100 bases from the coding region of adjacent genes. Sequence identity was calculated for the region encompassed by 200 bases upstream of the translation start codon, i.e. promoters and UTRs. We observed that coding regions across all genera and genes displayed sequence identity of 80% to 97%, whereas the non-coding regions were 45% to 79%. In keeping with our findings throughout this study employing the psbA 5′ UTR as an experimental model system we found that despite 95.0% identity in the coding region, identity in the psbA upstream region is 59% across all taxa (
To determine how the nucleotide sequence variability in this region affects the secondary structure of the UTR, we used RNAfold to generate two-dimensional structure predictions based on minimum Gibbs free energy (ΔG;
The pUC based L. sativa long flanking sequence vector was used to integrate foreign genes into the intergenic spacer region between the trnI and trnA genes as described previously (Ruhlman et al., 2007). The L. sativa native 16S ribosomal operon promoter (Prrn) and 3′ rbcL were amplified from the L. sativa plastid genome and used to regulate the expression of the aadA gene from the GGAG ribosome binding site for spectinomycin resistance. The aadA expression cassette was inserted into the spacer region between the trnl and trnA and resulted in pLsDV vector. The pLsDV NtCTB-Pins and pLsDV LsCTB-Pins plasmids were constructed by transferring the CTB-Pins expression cassette with N. tabacum and L. sativa psbA regulatory sequences to pLsDV. pLsg10 PAG was constructed by replacing CTB-Pins with pag in pLS CTB-Pins vector. The pLsDV LsPAG construct was made by cloning pag expression cassette with L. sativa psbA regulatory sequences to the pLsDV vector. The plasmids pLD VK1, pLD-5′UTR-CTB-Pins and pLS CTB-Pins used to generate Nt-Nt-PA, Nt-Nt-CP and Ls-g10-CP plants have been reported previously (
Plastid transformation of L. sativa and N. tabacum was carried out as described (Daniell et al., 2005; Ruhlman et al., 2007), with modifications of regeneration media as described above. Primary regenerants identified by PCR were subjected to an additional round of regeneration followed by rooting in selective media. Site specific integration and homoplasmy of the transplastome was confirmed by Southern hybridization with flanking sequence probes specific for L. sativa or N. tabacum. Generation and confirmation of homoplasmy for CTB-Pins lines Nt-Nt-CP, Ls-g10-CP, and PA line Nt-Nt-PA have been reported previously (Koya et al., 2005; Ruhlman et al., 2007). All newly generated transplastomic lines were found to be homoplasmic, containing no detectable wild type copies of the plastome (
Second generation (T1) transplastomic homoplasmic plants of L. sativa and N. tabacum expressing CTB-Pins or PA were grown in the greenhouse for 8-10 weeks. Leaves of different developmental stages were harvested at four time points during the light cycle to evaluate foreign protein accumulation. Expression of CTB-Pins was quantified by densitometric analysis of crude homogenates against known quantities of CTB standard (
Expression of PA in transplastomic N. tabacum and L. sativa was determined by ELISA of young, mature and older leaves. PA was mostly observed in the soluble fraction and therefore quantitation by ELISA was accurate. The maximum PA expression was observed in mature leaves compared to young or older leaves in all the lines examined. In mature leaves harvested at 6 pm from L. sativa and N. tabacum lines, PA accumulation reached up to 22.4% +/−1.0 and 29.6% +/−0.9 of total soluble protein (TSP) respectively, when PA expression was regulated by endogenous regulatory elements. In Nt-Ls-PA, the foreign protein represented 5.8% +/−0.1 of TSP in mature leaves at 6 pm, an 80% reduction in expression in N. tabacum (
Total RNA was isolated from different transplastomic lines and northern blots were prepared to examine the transcript populations generated from various regulatory elements. Blots were probed with radiolabeled coding regions for CTB-Pins (
Total RNA was prepared from fractions separated through sucrose gradients to evaluate polysome association of foreign gene transcripts in L. sativa transformants that express CTB-Pins from the endogenous psbA UTR (Ls-Ls-CP) versus the N. tabacum psbA 5′ UTR (Ls-Nt-CP). The distribution of ribosomal RNAs in gradient fractions was similar between these two lines. Northern blots of 12 fractions collected from the bottom of the gradient were probed with the radiolabeled, full-length coding sequence for CTB-Pins to localize transplastomic transcripts. Blots prepared from all fractions off the gradient for Ls-Ls-CP or LS-Nt-CP lines show that the CTB-Pins transcript present in all fractions was predominantly monocistronic, although the dicistron is readily detectable and in some fractions abundant (
Extracts of plastid stromal proteins were prepared for L. sativa and N. tabacum as described in methods. Labeled, full length psbA 5′ UTRs specific to each system were transcribed in vitro as were the unlabeled competitors. Reactions were separated in native gels to evaluate the affinity of native UTRs for stromal protein binding in the presence of up to 200-fold molar excess of competitor species. As shown in
To determine if the RBP interaction affected mRNA turnover in these studies, we synthesized labeled LspsbA 5′ UTR CTB-Pins transcripts in vitro, incubated them in either L. sativa or N. tabacum stromal extracts and extracted total RNA over time. RNAs were electrophoresed, gels were dried and exposed to film for densitometric analysis. We found the half life of the radiolabeled transcript to be 8.4 min in the homologous
L. sativa extract while incubation of the transcript in the N. tabacum stromal extracts reduced the half life to 2.3 min, a 3.7 fold difference in the rate of turnover (
To determine if CTB-Pins protein was differentially stabilized in transplastomic lines we conducted protein labeling with 35S and extracted leaf proteins at different time points throughout the chase period. Immunoprecipitation with CTB antibody was used to isolate the CTB-Pins protein away from the other labeled proteins translated during the pulse period.
The foregoing examples disclose a rapid and reproducible method of chloroplast transformation in L. sativa. A plant system having direct shoot regeneration, species specific vector with endogenous regulatory elements, optimized DNA delivery, age of the explants and culture conditions were crucial in optimization of L. sativa chloroplast transformation. We found that L. sativa cv. Simpson elite had the highest direct-shoot regeneration potential, with maximum number of shoots (4.3), among the five cultivars examined in this study. The ability to produce direct shoot regeneration from somatic cells varies among the different cultivars of L. sativa (Xinrun and Connor, 1992). The induction of direct-shoot was highly dependent on the auxin and cytokinin ratio. Auxin or cytokinin alone did not induce shoot in any of the cultivars. Whereas leaf incubated on medium supplemented with 0.1 μg/ml NAA+0.2 μg/ml BAP induced maximum number of shoots in all cultivars, confirming that the optimum level of auxin-cytokinin balance is essential to induce maximum number of shoots in all the cultivars examined. Two types of organogenesis are known. In “direct organogenesis” the explants undergo a minimum proliferation before forming an organ such as shoot or root, whereas in “indirect organogenesis” explants undergo an extensive proliferation before it develops into shoot or root (Ovecka et al., 2000) and often requires re-determination of differentiated cells (dedifferentiation; George. et al., 2008). The L. sativa chloroplast transformation system reported here is established through direct organogenesis and therefore it is more rapid than the indirect organogenesis established through callus reported previously (Kanamoto et al., 2006; Lelivelt et al., 2005). Regeneration and efficiency of this system is comparable to tobacco, the most efficient transplastomic system developed so far. Such a rapid and reproducible transformation system has already facilitated expression of several vaccine antigens or autoantigens in lettuce chloroplasts.
A survey of the literature demonstrates that there is considerable interest in chloroplast transformation, both in the established system of N. tabacum and in crop species that have been more recalcitrant to transformation by this approach. More than 20 reports describe the generation of transplastomic crop plants through the use of species-specific flanking regions to facilitate homologous recombination between the shuttle vector and the plastid genome. However, the expression cassettes used in these experiments more contain heterologous regulatory elements either from another plant species, usually N. tabacum. In this study we have utilized untransformed L. sativa and N. tabacum plants along with a suite of transplastomic lines to investigate potential mechanisms that underlie the variation we have observed in foreign protein accumulation resulting from the use of endogenous or heterologous regulatory elements. The findings presented herein show that these differences may be influenced at multiple levels including transcription, mRNA processing, mRNA stability, and translation of foreign gene products.
Relative abundance of foreign gene transcripts in total RNA extractions of transplastomic lines were examined and the greatest variation in the monocistron pools in the different CTB-Pins lines were identified. Sequence alignments reveal that there is 100% identity between L. sativa and N. tabacum in the region of the ribosomal operon P1 promoter required for full PEP activity (−64 to +17; Suzuki et al., 2003). This is supported by similar levels of dicistron accumulation in the L. sativa and N. tabacum lines where transcription of this mRNA species is driven by foreign or native Prrn. Although this observation was consistent for Ls-Ls-PA and Ls-g10-PA lines, in Nt-Ls-PA, which carries the L. sativa Prrn, both the dicistron and monocistron were reduced. The core promoter elements shown to be sufficient for developmental regulation of psbA transcription are 90% identical between L. sativa and N. tabacum (−42 to +9; Hayashi et al., 2003) Therefore it seems unlikely that the differences that were observed in the foreign transcript pool result directly from an inability to achieve PEP-mediated synthesis due to divergence in promoter sequence and structure in the heterologous system, i.e. Ls-Nt-CP.
Based on sequence comparisons, the greatest variability upstream of the D1 coding region lies within the proposed stem-loop structure of the 5′ UTR that has been shown to be involved in transcript stability and translation efficiency in land plants (Alexander et al., 1998; Eibl et al., 1999; Shen et al., 2001; Zou et al., 2003). Over the entire sample set this region has a 61% sequence identity, and for L. sativa and N. tabacum the identity is 54.2%. The results provided herein from polysome analyses indicated that foreign transcripts in L. sativa lines which carried the endogenous 5′ UTR were more abundant in the upper fractions giving some insight into differences in foreign protein accumulation in the various lines used in this study.
It has been shown previously that the majority of psbA transcripts are not polysome associated in Hordeum vulgare, Spinacia oleracea and N. tabacum (Klein et al., 1988; Minami et al., 1988) and that this population is likely stabilized by RBPs in the stroma (Nakamura et al., 1999, 2001). A number of studies have investigated the cis-elements within the land plant psbA 5′ UTR to elucidate sequences which are required for the association of RBPs, and the role of these interactions in transcript stability, processing and initiation of translation on plastid ribosomes. Using synthetic psbA 5′ UTRs with specific site mutations and internal deletions it was determined that the stem loop region was a dispensable element as its exclusion did not affect translation of the fused lacZ reporter used in one study. An AU rich element (AU box) was identified between RBS1 and RBS2 that together were required and sufficient to initiate translation and proposed a role for the AU box as the primary target sequence for the binding of trans-acting factors (Hirose and Sugiura, 1996). Sequence analysis, combined with the results from RNA EMSA, suggest that the AU box and adjacent RBS sites are not responsible for differences observed in the experimental system. Between L. sativa and N. tabacum there was sequence identity of 95% over this region (20 bp), the variation generated by a single base change in RBS2. It was found, however, that this region has diverged in a subset of species, including representatives from each of the clades in our analysis, which have variable TA (AU) insertions at the 3′ end of the AU box (
Subsequent analyses have suggested the presence of an endonucleolytic cleavage site that is protected upon the binding of protein factors. This site is localized to the predicted stem-loop region of the S. oleracea psbA 5′ UTR (−49/−48 relative to start of translation) and the binding interaction is sensitive to the secondary structure (Klaff et al., 1997; Alexander et al., 1998). Additional support for the role of the stem loop structure in stabilization of transcripts that include the psbA 5′ UTR is provided from transplastomic studies. Nicotiana tabacum plants were generated that express uidA (GUS) mRNA from chimeric genes where transcription was driven by Prrn with the full length endogenous psbA 5′ UTR (85 bases) or deletion variants upstream of the GUS coding region. In all experimental constructs mRNA abundance was reduced compared to the control despite transcription from identical promoters (Zou et al., 2003).
RNA EMSA assays using wild type stromal extracts demonstrated that the heterologous psbA 5′ UTR was not an effective competitor for binding factors that may be involved in transcript stability, and it was observed that the half life of the foreign transcript was reduced by up to 3.7 fold in the heterologous background. This strongly preferential binding of the endogenous UTR is attributed to the stem loop structural element as this is the primary region where significant sequence variation exists between L. sativa and N. tabacum. Conceivably in planta the ability of foreign gene transcripts equipped with heterologous UTR elements to compete for stabilization factors would be hampered by the presence of abundant endogenous psbA transcripts, leading to rapid turnover of foreign RNA species in the plastid.
The species-specific nature of protein factor binding to the psbA 5′ UTR has been further evaluated as a mechanism influencing foreign protein accumulation in transplastomic lines. We found that exchange of the full length UTR between L. sativa and N. tabacum resulted in a reduction in CTB-Pins and PA expression of at least 97% and 80%, respectively. This effect was consistent in young and mature leaves sampled at four time points. We did not detect a difference in the stability of CTB-Pins expressed from native or heterologous elements suggesting it was synthesis of the foreign protein, rather than degradation, which resulted in highly variable accumulation. CTB-Pins accumulated to much higher levels in older, but not senescent, leaves of Ls-Nt-CP plants but this could not compensate for the differences in overall expression as accumulation still showed a reduction of approximately 85% compared to N. tabacum plants with the endogenous UTR construct. PA lines did not accumulate foreign protein in older leaves to the same levels as CTB-Pins lines (
We estimate that CTB-Pins in fully expanded leaf tissue comprised 57% to 58% of the total leaf protein when harvested near the end of the light period and reached as high as 72%. The use of endogenous psbA 5′ UTR has led to the accumulation of numerous gene products in transplastomic N. tabacum (Verma et al., 2008), including proteins that had been previously unattainable at satisfactory levels using this technology (Fernandez-San Millan et al., 2003; Dhingra et al., 2004; Singh et al., 2008). We have now generated transplastomic L. sativa plants that accumulate abundant foreign protein. While the expression of first therapeutic protein produced in L. sativa was accomplished using the N. tabacum Prrn g10 system for expression of the gene of interest and the selectable marker (Ruhlman et al., 2007), the implementation of L. sativa specific regulatory elements has contributed to the development of a highly reproducible transformation system that generates transplastomic L. sativa plants expressing foreign proteins to high levels.
There are a great many factors to consider when designing transformation vectors for the generation transplastomic lines, particularly when the target is a new species for which there is no standardized approach. The use of species specific integration sequences (Verma and Daniell, 2007), codon optimization for plastid expression (Tregoning et al., 2003) and the inclusion of N-terminal stabilization sequences have been essential to the development of novel transplastomic lines (Ye et al., 2001; Leelavathi and Reddy, 2003). The emergent study of pentatricopeptide repeat (PPR) proteins in addition to well established research on RBPs have revealed that the interactions between protein factors and their cognate RNA sequences is highly specific. Our evaluation of UTR sequences from taxonomically diverse species for genes representing the various functional groups found in plastids combined with our experimental findings using the psbA 5′ UTR in particular argues for the use of species-specific regulatory elements for significant accumulation of foreign protein in transplastomic plants. This underscores the need for determining complete chloroplast genome sequences of crop species. It is equally important to optimize regeneration and tissue culture conditions by examining growth hormone requirements to achieve reproducible and rapid transformation of crop species.
Optimization of Direct Shoot Regeneration
Seeds of L. sativa cultivars (New England Seed Co., Hartford, Conn.) were disinfected in a solution of 30% commercial bleach with 0.01%, Tween-20 for 5 min, rinsed five times in sterile water and placed on half strength Murashige and Skoog (MS; Murashige and Skoog, 1962) medium solidified with 0.6% Phytablend® (Caisson, N. Logan, Utah). After 21 days, fully expanded first leaves were dissected into 0.8 cm 2 and placed adaxial side on the medium. The culture medium was composed of MS basal salts, 3% sucrose and 0.6% Phytablend. Plant growth regulators were added to the medium as shown in table 1 and pH was adjusted to 5.8 prior autoclaving. Cultures were maintained at 26±2° C. at 40 μE m2 s−1 photon density; 16 hrs light/8 hrs dark.
Genomic Analyses
Sequences of IGS upstream from genes representing different functional groups were extracted from complete plastid genomes on GenBank for 20 species representing most major clades of angiosperms (see supplemental tables 1 & 2 for taxa and genes). Alignments were anchored by the inclusion of 100 bases from the coding region of adjacent genes. Sequences were aligned using MUSCLE (Edgar, 2004), followed by manual adjustment in Geneious (http://www.geneious.com/). Sequence identity was calculated for the region encompassed by 200 bases upstream of the translation start codon. For comparison sequence identity was also determined for the aligned coding regions. Geneious was used to conduct motif searches for functional domains such as promoter and cis translation elements and to calculate sequence identities for selected regions.
Vector Construction
The pUC-based L. sativa long flanking plasmid (pLS-LF) was used to integrate foreign genes into the intergenic spacer between tRNA-Ile and tRNA-Ala genes of the plastome IR. Details of this vector, the g10 CTB-Pins expression cassette and the pLD-CtV-5CP N. tabacum transformation vector are published (Ruhlman et al., 2007). The L. sativa native Prrn and 3′ rbcL for the expression of aadA from a GGAG RBS were amplified using total genomic L. sativa DNA with sequence specific primers and assembled in pBSSK+ vector. The aadA cassette was subcloned into pLS-LF at PvulI site and the resulting vector is named as pLsDV. The L. sativa native psbA 5′ and 3′ UTR were PCR amplified and cloned in pBSSK+vector to make an intermediate vector with multiple cloning site in between psbA 5′ and 3′ UTR resulting in pDVI-1. The CTB-Pins sequence and pag sequence was cloned at NdeI and XbaI site of pDVI-1 vector. The CTB-Pins and pag expression cassette with psbA 5′ and 3′ UTR was released by digestion with SalI and NotI and ligated into the pLsDV vector. A transformation cassette for the generation of transplastomic L. sativa plants that express CTB-Pins from N. tabacum psbA 5′ and 3′ UTR was assembled by digestion of the pLD-CtV-5CP plasmid with SalI and XbaI to release the CTB-Pins coding region plus the N. tabacum psbA 5′ UTR. This fragment was ligated into a pUC intermediate plasmid upstream of the N. tabacum psbA 3′ UTR. Nucleotide sequence of the intermediate plasmid pUC-NtUTR-CTB-Pins was confirmed. The cassette was released by digestion with SalI and SnaBI and ligated into the modified pLsDV vector digested by SalI and EcoRV. The pag coding sequence was released from pLD VK (Koya et al., 2005) plasmid by digesting with NdeI and NotI and cloned in pLsg10 CTB pins vector. All cloning steps were carried out in E. coli according to the methods of Sambrook and Russel (2001).
Bombardment and Selection of Transplastomic Lettuce
Seeds of L. sativa cv. Simpson elite (New England Seed Co.) were surface sterilized MS media solidified with 6 g L−1 Phytablend® (Caisson). Young, fully expanded leaves ˜4 cm2 were placed adaxial side up on antibiotic free L. sativa regeneration (LR) media (Kanamoto et al., 2006). Leaves were bombarded with 0.6 μm gold particles (BioRad) coated with one of the plastid transformation vectors shown in
PCR Screening and Southern Blotting
Genomic DNA isolated from primary transformants was analyzed by PCR using primers 16SF (5′CAGCAGCCGCGGTAATACAGAGGA3′) and 3M (Singh et al., 2009). For Southern blotting, genomic DNA was isolated from young, in vitro grown leaves ground in liquid N2 with chilled, sterile mortar and pestle, and extraction was carried out using a QIAGEN DNeasy® Plant mini kit (#69104) according to the manufacturer's protocol. Five micrograms of total DNA was digested to completion with BgIII (g10) or AfIIII (Nt-UTR) for CTB-Pins lines and SmaI for all PA lines, the resulting fragments were separated on 0.8% TAE-agarose gels and transferred to nylon membranes by capillary action. Plastid flanking sequence probe (1.3 kb) was amplified by PCR from L. sativa genomic DNA. PCR product was column purified and labeled probe was generated by incubation with α-32P-dCTP and Ready-To-Go™ DNA Labeling Beads (GE Healthcare). Blots were pre-hybridized for one hour at 68° C. in QuikHyb® reagent (Stratagene, Cedar Creek, Tex.). Blots were hybridized for 1 hr 68° C., washed twice at 37° C. in 2×SSC (0.3 M sodium chloride, 30 mM sodium citrate, pH 7) and twice at 65° C. in 0.1×SSC. Radiolabeled blots were exposed to film on intensifying screens at −80° C. for 16 hrs.
Western Blot and Densitometric Analysis
Second generation (T1) CTB-Pins transplastomic L. sativa and N. tabacum were raised in the UCF greenhouse. Young, mature and older fully expanded leaves from ˜8 week old plants were harvested in August at 5 am, 10 am, 2 pm and 6 pm, ground in liquid N2 and stored at −80° C. Approximately 100 mg of leaf tissue was suspended in five volumes of protein extraction buffer (100 mM NaCl, 10 mM EDTA, 200 mM Tris-HCl pH 8, 0.1% Triton X-100, 100 mM DTT, 400 mM sucrose, 2 mM PMSF). Extractions were vortexed vigorously for 20 min at 4° C. prior to determination of total protein using Bio-Rad Protein Assay Reagent. Total leaf proteins along with 100, 200, 400 and 600 ng of purified bacterial CTB. (Sigma, St. Louis, Mo.) were separated by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes for immunoblotting, according to Kumar and Daniell (2004). Immunoblotting with anti-CT primary antibody (1:3, 500, Sigma) and HRP-conjugated goat anti-rabbit secondary antibody (1:4,000, Southern Biotech, Birmingham, AL) was employed for densitometric analysis. A SuperSignal® West Pico HRP Substrate Kit (Pierce, Rockford, Ill.) was used for detection of chemiluminescence signal by exposure to film. Tissue collected at the latest developmental stage was prepared as above and subjected to SDS-PAGE along with CTB standards ranging from 0.5 to 3 μg. Gels were stained with Coomassie blue and used for densitometric quantitation.
Estimation of PA Protein Using ELISA
T1 PA transplastomic L. sativa and N. tabacum were raised in the UCF greenhouse. Young, mature and older, fully expanded leaves from ˜8 week old plants were harvested at 6 am, 12 pm, 6 pm and 3 am, ground in liquid N2 and stored at −80° C. ELISA of leaf extract supernatant was performed in duplicate in a 96-Well EIA/RIA plate (Costar, Corning Inc. NY), along with purified PA as standard (courteously provided by Dr. Stephen H Leppla, NIH, Bethesda, Md.) The standard and samples were diluted in 15 mM Na2CO3, 35 mM NaHCO3, 3 mM NaN3, pH 9.6. PA standard ranging from 5 ng ml−1 to 400 ng ml−1 and sample dilutions of 1:10,000, 1:20,000 and 1:40,000 were loaded into the wells and incubated at 4° C. overnight. The plate was blocked with 3% fat-free milk in phosphate buffer saline and 0.05% Tween-20 (PBST), incubated at 37° C. for 1 hr and washed three times each with PBST and water. Primary monoclonal anti-PA (1:3,000) in PBST was loaded into wells and incubated at 37° C. for 1 hr. The wells were then washed with PBST and water as above. HRP-conjugated goat anti-rabbit (1:5, 000; Southern Biotech) was, incubated at 37° C. for 1 hr followed by washing with PBST and water. 100 μl of 3.3′, 5.5′-tetramethyl benzidine (TMB) substrate was loaded to each well and incubated at 24° C. for 10-15 min. The reaction was terminated by addition of 50 μl of 2N H2SO4. Plate was read at 450 nm using a plate reader (Model 680, Bio-Rad).
Plastid Isolation
Intact N. tabacum plastids were isolated according to Yukawa et al. (2007). Fully expanded green leaves (200 g) were collected from 4-5 week old greenhouse grown N. tabacum cv TN90. Leaves were homogenized in 50 g batches with 150 mL MCB1. Homogenates were filtered through 4 layers of cheesecloth then 2 layers of Miracloth (Calbiochem) and centrifuged for 5 min at 1,000×g in a Sorvall SS-34 fixed angle rotor at 4° C. Pellets were resuspended by gentle agitation with a soft paintbrush in 30 mL MCB1 and 5 mL was layered onto 25 mL Percoll (Sigma) gradients (20%-50%-80% in MCB1). Gradients were centrifuged for 10 min at 10,000×g in L-90K ultracentrifuge SW32-ti rotor; 2° C. The lower dark green band containing intact plastids was harvested, washed in three volumes MCB2. Plastids were collected by 1 min centrifugation at 600×g in Sorvall SS-34 at 4° C. Plastid pellet was resuspended in minimal volume of EMSA binding buffer (Alexander et al., 1998) with rigorous agitation to disrupt plastid envelopes. Membranes were sedimented by centrifugation in SS-34 rotor; 27,000×g for 15 min. Stromal extracts were adjusted to 15% glycerol, aliquoted and stored at −80° C.
Intact L. sativa plastids were isolated according to Gruissem et al. (1986). Fully expanded green leaves (200 g) of 6-8 week old hydroponically grown L. sativa cv longifolia were collected from the greenhouse and were homogenized in 50 g batches with 150 mL 1×GM buffer. Homogenates were filtered, centrifuged and pellets were resuspended as above in 1×GM buffer; 5 mL was layered onto 25 mL PCBF density gradients. Gradients were centrifuged for 20 min at 8,100×g. at 2° C. The lower dark green band containing intact plastids was harvested, washed in 2 volumes 1×GM buffer. Plastids were collected by 3 min centrifugation at 1,500×g at 4° C. Plastid pellet was resuspended in minimal volume of the above EMSA binding buffer and preserved as for N. tabacum
In vitro Transcription of Radiolabeled Transcripts
Plasmids (pBluescript SK+, Stratagene) containing the psbA 5′ UTR were digested with NdeI (L. sativa) or NcoI (N. tabacum) to generate linearized templates for T7 in vitro transcription using the MAXIscript® Kit (Ambion Inc, Austin Tex.) according to manufacturer's instructions. For mRNA turnover the L. sativa UTR-CTB-Pins plasmid was digested at the XbaI site situated at the 3′ end of CTB-Pins. For labeled UTR species uridine triphosphate (UTP) was replaced with 3.125 μM α-32P UTP (Perkin Elmer, Waltham, Mass.). Reaction products were separated by denaturing polyacrylamide and eluted following manufacturer's instructions. Following ethanol precipitation supernatants were discarded and RNA pellets were vacuum dried. Pellets were resuspended 50 μL nuclease-free water and quantified by spectrophotometry and liquid scintillation counting. Single use aliquots were prepared and stored at −80° C.
Electrophoretic Mobility Shift and mRNA Turnover
Competitive RNA EMSA was adapted from Alexander et al. (1998). Stromal extracts were thawed on ice and total protein content was determined using the Bio-Rad Reagent (Bio-Rad). Stromal proteins of N. tabacum (20 μg) or L. sativa (40 μg) were incubated with 5 fmoles of endogenous radiolabeled psbA 5′UTR, with or without unlabeled competitor psbA 5′ UTR. All reactions were supplemented with 0.5 μg uL−1 yeast tRNA (Ambion) to reduce non-specific binding and total volume was adjusted to 20 μL using EMSA binding assay buffer. Control reactions included no competition, competition with 50× molar excess of unlabeled native UTR and labeled probe only (no protein). Experimental reactions contained 50×, 100× and 200× molar excess of unlabeled non-native competitor UTR (i.e. N. tabacum protein with L. sativa UTR as competitor). Competitors were added 5 min prior to the labeled probe. Reactions were allowed to proceed for 15 min at 22° C. in the presence of labeled probe. 4 μL of 5× non-denaturing gel loading buffer was added (225 mM Tris-HCl, pH 6.8, 50% glycerol, 0.05% bromophenol blue) and reactions were separated through 8% polyacrylamide.
For degradation analysis of the labeled Ls-5′UTR-CTB-Pins transcript in stromal extracts the no competition control reaction described above was used. At the designated time points total RNA was isolated by phenol:chloroform as described below and extracts were separated by electrophoresis. Gels were vacuum dried and exposed to film for 6-12 hrs for analysis of binding reactions.
Polyribosome Association Assay and Northern Blotting
Polysome assay was adapted from Barkan et al., (1988). Green leaves from in vitro grown plants were ground to a fine powder in liquid N2. Approximately 300 mg of each sample was transferred to a 2 mL tube and vortexed with 1 mL extraction buffer (0.2 M Tris HCl pH 9, 0.2 M KCl, 35 mM MgCl2, 25 mM EGTA pH 8.3, 1% Triton X100, 2% polyoxyethylene-10-tridecylether) with 0.5 mg mL−1 heparin, 100 μg mL−1 chloramphicol, 25 μg mL−1 cyclohexamide. Homogenates were forced gently through glass wool packed in 3 mL syringe into microcentrifuge tube on ice and extracts were held on ice for 10 min. Extracts were centrifuged at 17,900×g for 5 min at 4° C. Supernatants were transferred to new tubes and 1/20th volume of 10% sodium deoxycholate was added. Control reactions were incubated with puromycin (3 mg mL−1) at 37° C. for 10 min. Reactions were held on ice for 5 min prior to centrifugation at 17 900×g for 15 min at 4° C. Supernatants (500 μL) were layered onto 15%-30%-40%-50% sucrose gradients in 10× salts (0.4 M Tris-HCl, pH 8, 0.2 M KCl, 0.1 M MgCl2) prepared in Beckman ultraclear ½×2 inch tubes (13×51 mm; 344057). One aliquot of supernatant from each sample was reserved for isolation of total RNA. Gradients were centrifuged at 4° C. in SW55-ti rotor for 65 min at 192,000×g. Fractions of approximately 500 μL were collected into microfuge tubes containing 50 μL of 5% SDS and 0.2 M EDTA pH 8, by puncturing gradient tubes with an 18 gage needle. One volume of phenol:chloroform:isoamyl (25:24:1) was added to each fraction and vortexed then centrifuged at 17,900×g for 5 min. The aqueous phase was transferred to a new tube, two volumes absolute ethanol was added and mixed by inversion. RNA was pelleted by centrifugation, supernatant was discarded and pellets were dried under vacuum. Pellets were resuspended in 30 μL Tris-EDTA. RNA sample buffer (80 mM MOPS, pH 7, 4mM EDTA, 0.9 M formaldehyde, 20% glycerol, 30.1% formamide, 5 mM sodium acetate, 0.25% bromophenol blue) was added and fractions were separated in formaldehyde-agarose gels under denaturing conditions. Gels were washed in RNase-free water and RNA was transferred to nylon membranes (Nytran SPC, Whatman Inc., Sanford Me.) by capillary action in 20×SSC. Membranes were rinsed in RNase-free water and fixed by UV crosslinking. The full length CTB-Pins coding region was used to generate α-32P-labeled, single stranded DNA probes according to the procedure described above for Southern blotting. Pre-hybridization and hybridization steps for polyribosome blots were carried out in Denhardt's buffer. Blots of total RNA extractions were hybridized in QuikHyb (Stratagene). For analysis of total transcripts, RNA was extracted using the Qiagen RNeasy®Mini Kit (Qiagen), and quantified by spectrophotometry. Total RNA, 2.5 μg, was prepared electrophoresed and blotted as described above.
Pulse-Chase Labeling and Immunopreciptation
Fully expanded leaves of L. sativa and N. tabacum transplastomic lines were cut into 5 mm2 explants under half strength MS media with a sharp, sterile blade. Explants, approximately 50 per sample, were placed in a vacuum apparatus containing 10 mL MS media with 0.05% Tween 20 and 1 mCi EXPRESS 35S protein labeling mix (PerkinElmer). Explants were infiltrated for 90 sec, transferred to a Petri dish along with the media and incubated in the light for 1 hr. Explants were removed from isotope media, rinsed with sterile water and transferred to the vacuum apparatus with fresh media without isotope containing 10 mM methionine. Infiltration was repeated and explants and media were transferred to a Petri dish for duration of chase period under 16 hrs light and 8 hrs dark in the growth chamber. At the time intervals shown in results, explants (5-6 each; ˜25 mg) were removed from the media, blotted on tissue paper and transferred to microcentrifuge tubes. Samples were frozen in liquid nitrogen and stored at −80° C. until the end of the chase period.
Samples were ground to a fine powder using a glass pestle with liquid N2, in tubes. Approximately eight volumes of homogenization buffer (200 mM Tris-HCl pH 8, 100 mM NaCl, 10 mM EDTA, 0.1% SDS, 0.05% Tween 20, 2 mM PMSF) was added to each sample followed by vortexing with a micropestle. Total protein in homogenates was quantified using the Bio-Rad Reagent. An equal amount of total protein was taken from each sample and diluted to 1 mL in PBS. Primary antibody (a-cholera toxin from rabbit, 1:500, Sigma) was added to each sample and tubes were placed on a rocker at 4° C. for 4-6 hrs. To each tube 40 μL protein A-agarose (Santa Cruz Biotech, Santa Cruz, Calif.) was added and samples were incubated on the rocker at 4° C. for 16 hrs. Samples were pelleted by pulse centrifugation and aspirated. Pellets were washed three times with 500 μL PBS. Final pellet was suspended in 50 μL electrophoresis sample buffer (90 mM Tris HCl pH 6.8, 20% glycerol, 2% SDS, 0.02% Bromophenol blue, 100 mM dithiolthreitol) and boiled for 2 min. Samples were centrifuged and half of the supernatant was used for scintillation counting, while the other half was analyzed by SDS-PAGE. SDS gels were dried and exposed to film to visualize immunoprecipitated, labeled protein.
In reviewing the detailed disclosure which follows, and the specification more generally, it should be borne in mind that all patents, patent applications, patent publications, technical publications, scientific publications, and other references referenced herein are hereby incorporated by reference in this application in order to more fully describe the state of the art to which the present invention pertains.
Reference to particular buffers, media, reagents, cells, culture conditions and the like, or to some subclass of same, is not intended to be limiting, but should be read to include all such related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material or composition is directed.
It is important to an understanding of the present invention to note that all technical and scientific terms used herein, unless defined herein, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. The techniques employed herein are also those that are known to one of ordinary skill in the art, unless stated otherwise. For purposes of more clearly facilitating an understanding the invention as disclosed and claimed herein, the following definitions are provided.
While a number of embodiments of the present invention have been shown and described herein in the present context, such embodiments are provided by way of example only, and not of limitation. Numerous variations, changes and substitutions will occur to those of skilled in the art without materially departing from the invention herein. For example, the present invention need not be limited to best mode disclosed herein, since other applications can equally benefit from the teachings of the present invention. Also, in the claims, means-plus-function and step-plus-function clauses are intended to cover the structures and acts, respectively, described herein as performing the recited function and not only structural equivalents or act equivalents, but also equivalent structures or equivalent acts, respectively. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the following claims, in accordance with relevant law as to their interpretation.
Agrostis stolonifera
Citrus sinensis
Coffea arabica
Cucumis sativus
Daucus carota
Eucalyptus globulus
Eucalyptus
Glycine max
Gossypium barbsfense
Gossypium hirsutum
Helianthus annuus
Hordeum vulgare
Lactuca sativa
Manihot esculenta
Nicotiana tabacum
Oryza nivara
Oryza sativa
Panax ginseng
Ginseng
Phaseolus vulgaris
Pinus koraiensis
Pinus thunbergii
Populus alba
Populus trichocarpa
Saccharum hybrid
Saccharum officinarum
Solanum
bulbocastanum
Solanum lycopersicum
Solanum tuberosum
Sorghum bicolor
Sorghum
Spinacia oleracea
Triticum aestivum
Vitis vinifera
Zea mays
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The teachings of all references cited herein are incorporated herein in their entirety to the extent they are not inconsistent with the teachings herein. U.S. Patent Publication 20090022705 is specifically incorporated herein by reference, which provides discussion on edible plants and molecular plant transformation techniques and sequence identity calculations.
This application is a continuation of U.S. Ser. No. 12/795,572 filed Jun. 7, 2010 which is related to U.S. Provisional Application No. 61/184,673 filed Jun. 5, 2009 and to which priority is claimed under 35 USC 119 and the entirety of the noted application is incorporated herein.
| Number | Date | Country | |
|---|---|---|---|
| 61184673 | Jun 2009 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12795572 | Jun 2010 | US |
| Child | 13865146 | US |
