# Patent

## Title
Compositions and methods for increasing transgene expression in the plastids of higher plants

## Abstract

Nucleic acid constructs comprising a highly efficient 5′ regulatory region for the expression of heterologous proteins from the plastids of higher plants are provided. Also provided are plant cells and transgenic plants comprising the same.

## Background
The present application is §371 application of
PCT/US2007/066859 filed 18 Apr. 2007 which claims priority to US Provisional Application No. 60/792,853 filed 18 Apr. 2006, the entire disclosure of each being incorporated by reference herein.


FIELD OF THE INVENTION
This invention relates to the fields of molecular biology and the expression of transgenes in the plastids of higher plants. More specifically, the invention provides DNA constructs, and vectors for enhancing the expression level of transgenes encoding proteins having commercial or therapeutic applications. Also provided are transgenic plants comprising such DNA constructs and vectors.
BACKGROUND OF THE INVENTION
Several publications and patent documents are cited throughout this application in order to better describe the state of the art to which this invention pertains. Each of these citations is incorporated by reference herein.
Potato tuber moth (Phthorimaea operculella, Lepidoptera, Gelechiidae) is one of the most destructive insect pests of potato with a pandemic distribution. In the field, the moths lay their eggs on the potato foliage and the larvae mine the foliage and the stems. Larvae attack the tubers through infected stems or may enter the tubers directly. Development of cultivars resistant to potato tuber moth through conventional breeding has not been successful because of lack of reliable resistance sources in potato germplasm. However, considerable degree of protection has been achieved by using insecticidal crystal proteins of the soil bacterium Bacillus thuringiensis (B.t.). Insecticidal crystal proteins are susceptible to UV damage necessitating frequent sprays on the standing crop. To overcome this, transgenic potato lines with tuber moth resistance have been developed by engineering cry1 class B.t. genes. Transgenic potato lines with variable level of PTM resistance have been obtained by expressing the native cry1Aa (Chan et al., 1996), cry1Ab (Jansens et al., 1995) and cry1Ac (Ebora et al., 1994) genes. Nuclear transformation with native cry genes in plants, however, results in very low levels of B.t. protein expression due to instability of prokaryotic transcripts in plant systems (Murray et al., 1991). Relatively high level of B.t. protein expression with better PTM control could be achieved by using codon modified and truncated cry1Ac9 (Beuning et al., 2001; Davidson et al., 2002), cry 1Ia1 (Mohammed et al., 2000; Douches et al., 2002), and a hybrid Bt toxin (SN19) gene consisting of domain I and III of cry1Ba and domain II of cry1Ia (Naimov et al., 2003). Gleave et al. cloned and sequenced a B.t. gene, later named cry9Aa2, from Bacillus thuringiensis var. galleriae (strain DSIR517) that showed strong insecticidal activity to P. operculella (LC50 80 ng/ml) (Gleave et al., 1992). The amino acid sequence of this new B.t. protein was significantly different from those belonging to Cry1 class and, therefore, it was placed under the new class of Cry9 (Crickmore et al., 1998). In their later work, Gleave et al. transformed tobacco with the native and the modified versions of the cry9Aa2 gene and found significant improvement in B.t., expression as well as PTM resistance in those expressing the truncated and codon modified versions (Gleave et al., 1998). It is apparent from the published work that sequence modification of cry nuclear genes is an essential requirement for achieving satisfactory levels of toxin expression and PTM control in transgenic plants.
SUMMARY OF THE INVENTION
In accordance with the present invention, the Cry9Aa2 crystal protein gene has been expressed in plastids and high levels of protein production obtained. Expression of transgenes in plastids involves placing the coding segment under control of prokaryotic-type plastid expression signals and incorporating the transgene into the plastid genome by homologous recombination events via plastid targeting sequences. There are 1,000 to 10,000 identical copies of the circular, double-stranded plastid genome  ̃150-kb in size. Uniform alteration of all genome copies is obtained by selective amplification of transformed copies and gradual loss of non-transformed copies during plant regeneration on a selective tissue culture medium (Bock & Khan, 2004; Maliga, 2004). Expression of B.t. insecticidal protein genes in the plastid genome was found useful to obtain high protein levels of cry1Ac (McBride et al., 1995), cry2Aa2 (Kota et al., 1999; De Cosa et al., 2001) and cry1Ia5 (Reddy et al., 2002) from native bacterial genes without a reported impact on plant fitness or imposing a yield penalty. We report here that expression of the cry9Aa2 gene also results in high expression levels,  ̃10% of the total soluble cellular protein and  ̃20% in the membrane fraction, and conferred resistance in feeding trials to potato tuber moth. However, unlike in the earlier cases, we also found that high Cry9Aa2 expression levels significantly delayed plant development, yet fully developed plants were indistinguishable from wild type.
Thus, a preferred embodiment of the invention comprises a nucleic acid construct comprising in the 5′ to 3′ direction, a 5′ nucleic acid sequence comprising a 49 nucleotide segment of the native cry9aA2 5′-UTR; b) a nucleic acid sequence encoding a heterologous protein or polypeptide of interest; and c) a transcription termination region, said construct optionally further comprising a nucleic acid encoding a selectable marker. The construct may optionally comprise flanking homologous sequences obtained from the plastid genome which facilitate homologous recombination into said genome. In a particularly preferred embodiment, the 49 nucleotide segment comprises SEQ ID NO: 4.
The constructs of the invention can also comprise sequences encoding heterologous proteins of interests. Such proteins include, without limitation, antibodies, hormones, interferons, bacterial toxins, viral proteins, cytokines, proinsulin, antimicrobial peptides, insecticidal proteins, and enzymes suitable for the production of polyhydroxybutyrate (polyhydroxyalkanoate) polymers. Also included in the invention are plant cells and transgenic plants obtained therefrom comprising the constructs described above.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Introduction of the cry9Aa2 gene into the tobacco plastid genome. (A) Map of the plastid-targeting region in plastid vector pPRV312L and cry9Aa2 vectors pSKC84 (Cry9Aa2 not tagged) and pSKC85 (Cry9Aa2 c-myc tagged) and of the cognate regions in the wild-type (wt) and transplastomic plants. Map positions are shown for: the cry9Aa2 gene; aadA, the selectable spectinomycin resistance gene; rrn16, trnI and trnA plastid genes. Wavy lines symbolize transcripts; vertical arrows above the line mark processed ends; triangles indicate loxP sites; RBS, marks position of ribosome binding site. Map position of the 2.1-kb EcoRI-HindIII probe is also shown. (B) DNA gel blot confirms cry9Aa2 integration in the tobacco plastid genome. Data are shown for transplastomic lines transformed with plasmids pSKC84 and pSKC85 and the wild-type parental line. BstEII digested total cellular DNA was probed with the 2.1-kb EcoRI-HindIII ptDNA fragment to detect the wild type (4.6 kb) and transgenic (5.9 kb and 2.2 kb) BstEII fragments.
FIG. 2. Testing mRNA accumulation by probing total cellular RNA with (A) cry9Aa2 (1.3-kb SwaI fragment), (B) aadA (0.8-kb NcoI-XbaI fragment) and (C) cytoplasmic 25S rRNA (loading control) probes.
FIG. 3. Cry9Aa2 protein accumulation in tobacco leaves. (A) Soluble (25 μg per lane) and membrane protein (30 μg per lane) fractions were separated by SDS-PAGE and stained with Coomassie blue R250. The position of Cry9Aa2 and the large (LSU) and small (SSU) rubisco subunits are marked by arrows. (B) The c-myc antibody recognizes the tagged Cry9Aa2 protein in Nt-pSKC85 extracts.
FIG. 4. The transplastomic Nt-pSKC84 and Nt-pSKC85 plants. (A) Expression of Cry9Aa2 gene delays development of transplastomic plants. Shown are one-month old seedlings. (B) Potato tuber moth bioassay on detached transplastomic tobacco leaves expressing the Cry9Aa2 crystal protein. All larvae died on the leaves of transplastomic tobacco lines Nt-pSKC84-19CA and Nt-pSKC85-5BA.



DETAILED DESCRIPTION OF THE INVENTION
We report here the control of potato tuber moth (Phthorimaea operculella) by incorporating a truncated Bacillus thuringiensis cry9Aa2 gene in the plastid genome. Plasmids pSKC84 and pSKC85 are derivatives of a new polycistronic plastid transformation vector, pPRV312L, that carries spectinomycin resistance (aadA) as a selective marker and targets insertions in the trnI-trnA intergenic region. The Cry9Aa2 N-terminal region (82.1 kDa; 734 amino acids) was expressed in a cassette, which consists of 49 nucleotides of the cry9Aa2 leader and the 3′-untranslated region of the plastid rbcL gene (TrbcL), and relies on readthrough transcription from the plastid rRNA operon. In a tobacco leaf bioassay, expression of Cry9Aa2 conferred resistance to potato tuber moth. In accordance, the Cry9Aa2 insecticidal protein accumulated to high levels,  ̃10% of the total soluble cellular protein and  ̃20% in the membrane fraction. However, high-level Cry9Aa2 expression significantly delayed plant development. Thus, a practical system to control potato tuber moth by Cry9Aa2 expression calls for down-regulation of its expression.
The following definitions are provided to facilitate an understanding of the present invention:
Heteroplastomic refers to the presence of a mixed population of different plastid genomes within a single plastid or in a population of plastids contained in plant cells or tissues.
Homoplastomic refers to a pure population of plastid genomes, either within a plastid or within a population contained in plant cells and tissues. Homoplastomic plastids, cells or tissues are genetically stable because they contain only one type of plastid genome. Hence, they remain homoplastomic even after the selection pressure has been removed, and selfed progeny are also homoplastomic. For purposes of the present invention, heteroplastomic populations of genomes that are functionally homoplastomic (i.e., contain only minor populations of wild-type DNA or transformed genomes with sequence variations) may be referred to herein as “functionally homoplastomic” or “substantially homoplastomic.” These types of cells or tissues can be readily purified to a homoplastomic state by continued selection.
Plastome refers to the genome of a plastid.
Transplastome refers to a transformed plastid genome.
Transformation of plastids refers to the stable integration of transforming DNA into the plastid genome that is transmitted to the seed progeny of plants containing the transformed plastids. Alternatively, transformation may also include the introduction and transient expression of heterologous DNA into the plastid or nuclear genomes.
Selectable marker gene refers to a gene that upon expression confers a phenotype by which successfully transformed plastids or cells or tissues carrying the transformed plastid can be identified.
Transforming DNA refers to homologous DNA, or heterologous DNA flanked by homologous DNA, which when introduced into plastids becomes part of the plastid genome by homologous recombination.
An alternative type of transforming DNA refers to a DNA which contains recombination site sequences for a site-specific recombinase or integrase. Insertion of this type of DNA is not dependent of the degree of homology between the transforming DNA and the plastid to be transformed but rather is catalyzed by the action of the recombinase or integrase on the first and second recombination sites.
“Operably linked” refers to two different regions or two separate genes spliced together in a construct such that both regions will function to promote gene expression and/or protein translation.
“Nucleic acid” or a “nucleic acid molecule” as used herein refers to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form. In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5′ to 3′ direction. With reference to nucleic acids of the invention, the term “isolated nucleic acid” is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated. For example, an “isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.
When applied to RNA, the term “isolated nucleic acid” refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues). An isolated nucleic acid (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.
The term “functional” as used herein implies that the nucleic or amino acid sequence is functional for the recited assay or purpose.
The phrase “consisting essentially of” when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID No:. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.
A “vector” is a replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element. Vectors, expression cassettes and methods suitable for the generation of transplastomic plants are described in U.S. Pat. Nos. 6,624,296, 6,472,586, 6,388,168, 6,376,744, 6,297,054, 5,877,402, and 5,451,513, by Maliga et al., the disclosures of which are incorporated by reference herein.
An “expression operon” refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), elements which regulate mRNA stability, processing and translation, terminators, and the like, and which facilitate the production of a polypeptide coding sequence in a host cell or organism. Such expression signals may be combined such that production of said polypeptide occurs transiently or is produced stably over the life of the cell.
The term “oligonucleotide,” as used herein refers to primers and probes of the present invention, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide.
The term “probe” as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method.
The term “primer” as used herein refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis. When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH, the primer may be extended at its 3′ terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield an primer extension product. The primer may vary in length depending on the particular conditions and requirement of the application.
Amino acid residues described herein are preferred to be in the “L” isomeric form. However, residues in the “D” isomeric form may be substituted for any L-amino acid residue, provided the desired properties of the polypeptide are retained. All amino-acid residue sequences represented herein conform to the conventional left-to-right amino-terminus to carboxy-terminus orientation The term “tag,” “tag sequence” or “protein tag” refers to a chemical moiety, either a nucleotide, oligonucleotide, polynucleotide or an amino acid, peptide or protein or other chemical, that when added to another sequence, provides additional utility or confers useful properties, particularly in the detection or isolation, to that sequence.
The terms “transform”, “transfect”, “transduce”, shall refer to any method or means by which a nucleic acid is introduced into a cell or host organism and may be used interchangeably to convey the same meaning. Such methods include, but are not limited to, transfection, electroporation, microinjection, PEG-fusion, biolistic bombardment and the like.
“Agroinfiltration” refers to Agrobacterium mediated DNA transfer. Specifically, this process involves vacuum treatment of leaf segments in an Agrobacterium suspension and a subsequent release of vacuum which facilitates entry of bacterium cells into the inter-cellular space.
“T-DNA” refers to the transferred-region of the Ti (tumor-inducing) plasmid of Agrobacterium tumefaciens. Ti plasmids are natural gene transfer systems for the introduction of heterologous nucleic acids into the nucleus of higher plants. Binary Agrobacterium vectors such pBIN20 and pPZP22 (GenBank Accession Number 10463) are known in the art.
A “plastid transit peptide” is a sequence which, when linked to the N-terminus of a protein, directs transport of the protein from the cytoplasm to the plastid.
A “clone” or “clonal cell population” is a population of cells derived from a single cell or common ancestor by mitosis.
A “cell line” is a clone of a primary cell or cell population that is capable of stable growth in vitro for many generations.
The materials and methods set forth below were utilized in the performance of Example I.
Construction of Transformation Vectors
Plastid transformation vector pPRV312L targets insertions in the trnI/trnA intergenic region in the plastid ribosomal RNA operon (GenBank Accession Number DQ489715, SEQ ID NO: 1). Vector pPRV312L is a pUC118 plasmid derivative in which the PvuII fragment was replaced with the SmaI-HindIII tobacco ptDNA fragment (nucleotides 104,093-106,202; GenBank Accession No. Z00044) (Shinozaki et al., 1986). The polycloning site and marker gene were introduced as a PvuII-SacI (blunted with T4 DNA polymerase) fragment. The aadA marker gene is expressed in a cassette consisting of the PrrnLatpBDB promoter (Kuroda & Maliga, 2001a) and TpsbA, the 3′-UTR of psbA gene (Shinozaki et al., 1986). The aadA gene is flanked by the P1 phage loxP sites to facilitate its excision by the CRE site-specific recombinase (Corneille et al., 2001; Lutz et al., 2006).
Plasmid pNZA10 carrying the cry9Aa2 gene (GenBank Accession number X58534) was obtained from the Bacillus Genetic Stock Center, The Ohio State University, Columbus, Ohio. First the HincII/BamH1 fragment (2.2 Kbp) encoding 49 nucleotides of the leader sequence and the N-terminal half of the cry9Aa2 gene was excised from plasmid pNZA10 and sub-cloned into a pBluescriptKS (Stratagene, La Jolla, Calif.) plasmid. The C-terminally truncated cry9Aa2 gene was converted into an EcoRI-XbaI fragment for expression in pPRV312L. The XhoI site upstream of the HincII was converted into an EcoRI site by blunting and linker ligation and an in-frame stop codon was introduced in a BamHI-XbaI linker (5′-GGATCCAtaattctaga-3′). The modified cry9Aa2 gene was excised as an EcoRI-XbaI fragment (SEQ ID NO: 2) and cloned in a plasmid pPRV312L derivative, which carried the 3′UTR of the rbcL gene (TrbcL; XbaI-HindIII fragment) to yield transformation vector pSKC84. Plastid vector pSKC85 is similar to pSKC84, except that it has a C-terminal c-myc tag (amino acids 410-419; EQKLISEEDL; SEQ ID NO: 4) (Kolodziej & Young, 1991) introduced in a BamHI-XbaI fragment (5′-GGATCCgaacaaaaactcatttctgaagaagacttgtgattctaga-3′; SEQ ID NO: 5) with the stop codon. The DNA sequence of the EcoR1-Xba1 fragment in plasmid pSKC85 is SEQ ID NO: 3.
Plastid Transformation
DNA for plastid transformation was prepared using the QIAGEN Plasmid Maxi Kit (QIAGEN Inc., Valencia, Calif.). Transforming DNA was introduced into leaf chloroplasts on the surface of tungsten particles (1 μm) using the Du Pont PDS1000He Biolistic gun (Svab & Maliga, 1993). Transplastomic plants were selected on RMOP medium containing 500 mg/L spectinomycin dihydrochloride. The transgenic plants were grown on MS (Murashige-Skoog) medium (Murashige & Skoog, 1962) containing 3% (w/v) sucrose and 0.6% (w/v) agar in sterile culture condition. A uniform population of transformed plastid genome copies was confirmed by DNA gel blot analysis after digestion with the BstEII restriction enzyme. Double-stranded DNA probes were prepared by random-primed 32P-labeling using the Ready-To-Go DNA Labeling Beads (Amershem Pharmacia Biotech, Piscataway, N.J.). The probe was the trnI-trnA plastid targeting region, encoded in an EcoRI-HindIII ptDNA fragment.
RNA Gel Blot Analysis
For RNA gel blot analysis (Silhavy & Maliga, 1998) 5 μg total cellular RNA was loaded per lane. Probes were prepared by random-primed 32P-labeling (see above). The cry9Aa2 and aadA probes were prepared using SwaI and NcoI-XbaI coding region fragments, respectively.
SDS-PAGE and Immunoblotting
Leaves for protein extraction were taken from greenhouse plants. To obtain total soluble leaf protein, about 200 mg leaf was homogenized in 0.1 ml buffer containing 50 mM Hepes/KOH (pH 7.5), 10 mM potassium acetate, 5 mM magnesium acetate, 1 mM EDTA, 10 mM DTT and 2 mM PMSF. Insoluble material from the soluble fraction was removed by centrifugation. The insoluble material was solubilized by adding 0.1 ml solubilization buffer containing 50 mM Hepes/KOH (pH 7.5), 2% lithium dodecyl sulfate and heating for 10 minutes at 95° C. The insoluble material was then removed by centrifugation. Soluble protein concentrations were determined by the Bradford Protein Assay Reagent kit (Bio-Rad, Hercules, Calif.); membrane proteins were quantified with the bicinchoninic acid (BCA) method (Pierce, Rockford, Ill.). The protein in the Comassie Blue stained soluble extracts was quantified with Alpha Innotech (San Leandro, Calif.) Alphaimager IS-2200 using the 1D-Multi Lane densitometry. Immunoblot analysis of Cry9Aa2 accumulation was carried out as described (Carrer et al., 1993) using commercial c-Myc antibody purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.).
Insect Bioassay
First, a homogenous, laboratory population of the potato tuber moth was established (Raman & Palacios, 1982). For the detached leaf bioassay fully expanded, young leaves were excised with a sharp blade and placed singly in sterile tissue culture plate on a moist filter paper disk. Five neonate larvae were released on each leaf. Following their incubation at 26° C. for 5 days, the leaf damage and feeding area was recorded. Five leaves were used for each transplastomic line and the experiment was repeated twice.
Results
Construction of Transplastomic Tobacco Plants with Cry9Aa2 Genes
The engineered cry9Aa2 gene was introduced into the plastid genome in the pPRV312L plastid vector, which targets insertions in the trnI-trnA intergenic region located between the rrn16 and rrn23 genes in the plastid rRNA operon. The pPRV312L vector carries a selectable spectinomcyin resistance (aadA) gene flanked by lox-sites for marker gene excision, and a multiple cloning site for passenger genes (FIG. 1A). For expression in plastids, 48 nucleotides of the cry9Aa2 leader and a DNA segment encoding 734 N-terminal amino acids (82.1 kDa) was linked to the 3′-untranslated region of the plastid rbcL gene (TrbcL) and cloned upstream of the selective marker (aadA) (FIG. 1A). The promoter-less cry9Aa2 construct in this vector relies on readthrough transcription from the plastid rRNA operon promoter. Plastid transformation vector pSKC84 encodes the Cry9Aa2 peptide. In vector pSKC85 the Cry9Aa2 C-terminus was fused with a c-myc tag to enable detection by the commercial c-myc antibody. Transplastomic plants were obtained by bombardment of 20 leaves with plasmids pSKC84 and pSKC85, which yielded 26 and 21 spectinomycin resistant clones; of these plastid transformation was confirmed in 13 and 12 clones, respectively. Uniform transformation of plastid genomes was verified by DNA gel blot analysis (FIG. 1B). Plants derived from an independent transformation event are designated by a serial number; letters distinguish plants regenerated from the same clonal event; multiple letters indicate successive cycles of plant regeneration.
Expression of cry9Aa2 Genes in Chloroplasts
RNA gel blot analysis was carried out to test cry9Aa2 mRNA accumulation. The cry9Aa2 transgene is transcribed from the rrn operon promoter (Prrn). Probing of the RNA blot with the cry9Aa2 coding segment revealed mRNAs 2.4- and 2.5-kb in size (FIG. 2). We have observed accumulation of two mRNA species differing with 0.1-kb in size when TrbcL was combined with a loxP site, which forms a 13-nt stem-loop structure (Tungsuchat et al., 2006). Therefore, we assume that the 5′-end of the transcript was generated by maturation of trnI and the 3′-end by processing within the TrbcL or at the loxP-site. In RNA samples from young leaves additional, partially processed mRNA species are visible, which are processing intermediates of the rrn operon transcript (FIG. 1A). Degradation of cry9Aa2 mRNA is apparent in each of the samples.
The selective marker aadA is transcribed from two promoters: its own promoter (PrrnLatpB+DS) and the native rrn operon promoter upstream of rrn16. Probing with aadA revealed two  ̃1.1-kb monocistronic messages, which did not separate on the blot shown in FIG. 2. These transcripts derive from the aadA gene promoter and from processing of the rrn operon readthrough transcript.
We tested protein accumulation by separating leaf protein extracts in SDS-PAGE. Staining with Comassie Blue revealed a novel band,  ̃82 kDa in size (FIG. 3A). The novel protein was present in both soluble and membrane protein fractions of transgenic plants. Antibody to the c-myc tag in Nt-pSKC85 plants confirmed the identity of the novel band as a Cry9Aa2 insecticidal protein. The protein in the Comassie Blue stained soluble extracts was quantified with Alpha Innotech Alphaimager IS-2200 using the 1D-Multi Lane densitometry. The Nt-pSKC84 and Nt-pSKC85 leaves contained comparable amounts of Bt protein,  ̃10% of total soluble protein (FIG. 3A). Immunoblot analysis showed that the Cry9Aa2 protein concentration in the membrane fraction was higher,  ̃20% (FIG. 3B).
Potato Tuber Moth Bioassay on Tobacco Leaves
Transgenic plants have been transferred to the greenhouse where they flowered and produced seed. When grown from seed, development of the Nt-pSKC84 and Nt-pSKC85 transgenic plants was significantly delayed and the young leaves had a pale green color (FIG. 4A). However, the older leaves were normal green and plants eventually reached maturity, at which stage they were indistinguishable from wild-type plants. Out of the 12 and 13 independently transformed clones (see above) plants from four lines were studied in greater detail in the greenhouse: Nt-pSKC84-19CA, Nt-pSKC84-16DA and Nt-pSKC85-5BA and Nt-pSKC85-6BB. Seed progeny of each of the lines behaved similarly. Since several, independently transformed lines have the same phenotype, we believe, that the delay in plant development is due to high levels of Cry9aA2 protein accumulations. Furthermore, based on experience with tobacco plants regenerated from hundreds of independently derived transplastomic lines (Bock & Khan, 2004; Maliga, 2004) we are very certain that the delay in plant development is not caused by the transformation method.
Detached leaves of Nt-pSKC84 and Nt-pSKC85 tobacco plants expressing the cry9Aa2 gene have been tested for insecticidal activity against neonate potato tuber moth larvae (FIG. 4B). Transplastomic lines gave complete control of leaf mining by potato tuber moth larvae: 100% larval mortality was observed within 48-72 hrs, whereas no mortality was seen on the control plants for at least 120 hours.
Discussion
The pPRV312L Plastid Vector
The new vector pPRV312L has a multiple cloning site and loxP-flanked (L) aadA marker gene for excision by the CRE recombinase (Corneille et al., 2001; Hajdukiewicz et al., 2001; Lutz et al., 2006). Vector pPRV312L shares the feature of an excisable marker gene with vector pPRV110L (GenBank Accession No. DQ211347) that targets insertions in the trnV-rps12 intergenic region (Lutz et al., 2006). Vector pPRV312L in this study targets insertions between the trnl/trnA genes in the plastid rrn operon. The trnl/trnA intergenic region has been used for insertion of heterologous genes; reviewed in (Daniell et al., 2005). Genes of interest inserted at this site thus far always had their own promoter. Significant levels of protein expression have been obtained only from the rRNA operon (rrn) promoter and the promoter of the psbA gene (Maliga, 2003; Daniell et al., 2005). Expression of multiple genes from polycistronic mRNAs is a more desirable approach, reducing the requirement for promoters. We have shown already that transcriptional fusion with the rbcL mRNA yields significant levels of protein expression (Staub & Maliga, 1995). Expression of the gene of interest in our vector pPRV312L relies on readthrough transcription from the strong rrn operon promoter. High-level accumulation of Cry9aA2 indicates that transcription from an upstream promoter and translation from a processed 5′-UTR is sufficient for protein expression. In plastids, posttranscriptional gene regulation is important and high-level protein accumulation is dependent on the choice of 5′-UTR (Kuroda & Maliga, 2001 a; Kuroda & Maliga, 2001b); for review see (Maliga, 2003). Thus, the 49 nucleotide B.t. cry9aA2 5′-UTR is a leader that promotes high-level protein expression in chloroplasts. The 49 Bt nucleotide sequence is: 5′-AACCCAAATAATGTTTTAAAATTTTAAAAATAA TGTAGGAGGAAAAATT-3′; (SEQ ID NO: 6). DNA sequences encoding the processed 5′ UTR and translation initiation codon (ATG), including trnl-trnA intron sequences and the polycloning site, are provided in SEQ ID NO: 4. The B.t. cry2Aa2 leader also promotes high-level protein expression (De Cosa et al., 2001). The 49-nucleotide cry9aA2 leader sequence reported here is unrelated to the cry2Aa2 leader and lacks the upstream open reading frames, which are present in the cry2Aa2 operon construct.
We used here a 49 nucleotide segment of the native cry9aA2 5′-UTR, which also promotes high levels of translation. However, mRNA degradation is also apparent on the RNA gel blot. This may be caused by cry9aA2 sequences, which target mRNAs for degradation.
Although the native rrn operon transcript is large (includes rrn16, trnI, trnA, rrn23, rrn4.5, rrn5 genes)(Strittmatter & Kossel, 1984), the large precursor is efficiently processed (Kishine et al., 2004), creating the 5′-UTR of cry9aA2 mRNA. Normally, transcription termination and/or processing of mRNAs within the TrbcL segment used here is inefficient, yielding significant amounts of dicistronic transcripts due to TrbcL readthrough (Serino & Maliga, 1997; Kuroda & Maliga, 2001a; Tregoning et al., 2003). Interestingly, in this case dicistronic  ̃3.5-kb cry9aA2-aadA mRNAs were absent in mature leaves, suggesting efficient transcription termination and/or mRNA processing due to having loxP downstream of TrbcL (Tungsuchat et al., 2006).
Insect Biocontrol with Plastid-Expressed Cry9Aa2
Because of its strong insecticidal activity against larvae (LC50=80 ng per ml diet for 5 days), Cry9Aa2 Bt insecticidal protein is highly desirable for the biocontrol of potato tuber moth (Gleave et al., 1998). Lethality from a codon-modified nuclear gene yielded at least 75% larval mortality in nine days. Larval mortality caused by expression of Cry9Aa2 in plastids in our study was significantly higher, 100% within two-to-three days.
High-level expression (10% of soluble, 20% of membrane protein) of Cry9Aa2 protein came at a price of significantly delayed plant development. In earlier publications, plastid-expressed Bt insecticidal proteins also accumulated to relatively high levels in leaves: 5% cry1Ac (McBride et al., 1995), 3% (Kota et al., 1999) and 45.3% (De Cosa et al., 2001) cry2Aa2 and 3% cry1Ia5 (Reddy et al., 2002). Interestingly, no adverse affect of B.t. protein expression was reported on plant development in any of these studies. 100% larval mortality was observed even if the B.t. proteins were expressed at relatively modest (3%-5%) levels (McBride et al., 1995; Kota et al., 1999; Reddy et al., 2002). Thus, expression of Cry9Aa2 at significantly lower levels should be sufficient to achieve efficient insecticidal control. To create a useful cry9Aa2 construct one is facing the unusual task of down-regulating translation efficiency.
REFERENCES


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APPENDIX A





Sequence Information















SEQ ID NO: 1


DNA sequence of plastid transformation pPRV312L;


vector with floxed aadA in trnI/trnA intergenic


region; in pUC118 vector derivative (circular DNA)


pPRV3l2L.seq; GenBank DQ489715 Length: 6348


GGGCCTTGTA CACACCGCCC GTCACACTAT GGGAGCTGGC


 


CATGCCCGAA GTCGTTACCT TAACCGCAAG GAGGGGGATG


 


CCGAAGGCAG GGCTAGTGAC TGGAGTGAAG TCGTAACAAG


 


GTAGCCGTAC TGGAAGGTGC GGCTGGATCA CCTCCTTTTC


 


AGGGAGAGCT AATGCTTGTT GGGTATTTTG GTTTGACACT


 


GCTTCACACC CCCAAAAAAA AGAAGGGAGC TACGTCTGAG


 


TTAAACTTGG AGATGGAAGT CTTCTTTCCT TTCTCGACGG


 


TGAAGTAAGA CCAAGCTCAT GAGCTTATTA TCCTAGGTCG


 


GAACAAGTTG ATAGGACCCC CTTTTTTACG TCCCCATGTT


 


CCCCCCGTGT GGCGACATGG GGGCGAAAAA AGGAAAGAGA


 


GGGATGGGGT TTCTCTCGCT TTTGGCATAG CGGGCCCCCA


 


GTGGGAGGCT CGCACGACGG GCTATTAGCT CAGTGGTAGA


 


GCGCGCCCCT GATAATTGCG TCGTTGTGCC TGGGCTGTGA


 


GGGCTCTCAG CCACATGGAT AGTTCAATGT GCTCATCGGC


 


GCCTGACCCT GAGATGTGGA TCATCCAAGG CACATTAGCA


 


TGGCGTACTC CTCCTGTTCG AACCGGGGTT TGAAACCAAA


 


CTCCTCCTCA GGAGGATAGA TGGGGCGATT CGGGTGAGAT


 


CCAATGTAGA TCCAACTTTC GATTCACTCG TGGGATCCGG


 


GCGGTCCGGG GGGGACCACC ACGGCTCCTC TCTTCTCGAG


 


AATCCATACA TCCCTTATCA GTGTATGGAC AGCTATCTCT


 


CGAGCACAGG TTTAGCAATG GGAAAATAAA ATGGAGCACC


 


TAACAACGCA TCTTCACAGA CCAAGAACTA CGAGATCGCC


 


CCTTTCATTC TGGGGTGACG GAGGGATCGT ACCATTCGAG


 


CCGTTTTTTT CTTGACTCGA AATGGGAGCA GGTTTGAAAA


 


AGGATCTTAG AGTGTCTAGG GTTGGGCCAG GAGGGTCTCT


 


TAACGCCTTC TTTTTTCTTC TCATCGGAGT TATTTCACAA


 


AGACTTGCCA GGGTAAGGAA GAAGGGGGGA ACAAGCACAC


 


TTGGAGAGCG CAGTACAACG GAGAGTTGTA TGCTGCGTTC


 


GGGAAGGATG AATCGCTCCC GAAAAGGAAT CTATTGATTC


 


TCTCCCAATT GGTTGGACCG TAGGTGCGAT GATTTACTTC


 


ACGGGCGAGG TCTCTGGTTC AAGTCCAGGA TGGCCCAGCT


 


GCATTTAAAT GGCGCGCCGA ATTCGAGCTC GGTACCCGGG


 


GATCCTCTAG AGTCGACCTG CAGGCATGCA AGCTTGCGGC


 


CGCagtAGCT TATAACTTCG TATAGCATAC ATTATACGAA


 


GTTATagatc cGCTCCCCCG CCGTCGTTCA ATGAGAATGG


 


ATAAGAGGCT CGTGGGATTG ACGTGAGGGG GCAGGGATGG


 


CTATATTTCT GGGAGAATTA ACCGATCGAC GTGCaAGCGG


 


ACATTTATTT TaAATTCGAT AATTTTTGCA AAAACATTTC


 


GACATATTTA TTTATTTTAT TATTATGAGA ATCAATCCTA


 


CTACTTCTGG TTCTGGGGTT TCCACGgcta gtagcGAAGC


 


GGTGATCGCC GAAGTATCGA CTCAACTATC AGAGGTAGTT


 


GGCGTCATCG AGCGCCATCT CGAACCGACG TTGCTGGCCG


 


TACATTTGTA CGGCTCCGCA GTGGATGGCG GCCTGAAGCC


 


ACACAGTGAT ATTGATTTGC TGGTTACGGT GACCGTAAGG


 


CTTGATGAAA CAACGCGGCG AGCTTTGATC AACGACCTTT


 


TGGAAACTTC GGCTTCCCCT GGAGAGAGCG AGATTCTCCG


 


CGCTGTAGAA GTCACCATTG TTGTGCACGA CGACATCATT


 


CCGTGGCGTT ATCCAGCTAA GCGCGAACTG CAATTTGGAG


 


AATGGCAGCG CAATGACATT CTTGCAGGTA TCTTCGAGCC


 


AGCCACGATC GACATTGATC TGGCTATCTT GCTGACAAAA


 


GCAAGAGAAC ATAGCGTTGC CTTGGTAGGT CCAGCGGCGG


 


AGGAACTCTT TGATCCGGTT CCTGAACAGG ATCTATTTGA


 


GGCGCTAAAT GAAACCTTAA CGCTATGGAA CTCGCCGCCC


 


GACTGGGCTG GCGATGAGCG AAATGTAGTG CTTACGTTGT


 


CCCGCATTTG GTACAGCGCA GTAACCGGCA AAATCGCGCC


 


GAAGGATGTC GCTGCCGACT GGGCAATGGA GCGCCTGCCG


 


GCCCAGTATC AGCCCGTCAT ACTTGAAGCT AGACAGGCTT


 


ATCTTGGACA AGAAGAAGAT CGCTTGGCCT CGCGCGCAGA


 


TCAGTTGGAA GAATTTGTCC ACTACGTGAA AGGCGAGATC


 


ACCAAGGTAG TgGGCAAAga acaaaaactc atttctgaag


 


aagacttgtg agtctagcta gaGCGATCCT GGCCTAGTCT


 


ATAGGAGGTT TTGAAAAGAA AGGAGCAATA ATCATTTTCT


 


TGTTCTATCA AGAGGGTGCT ATTGCTCCTT TCTTTTTTTC


 


TTTTTATTTA TTTACTAGTA TTTTACTTAC ATAGACTTTT


 


TTGTTTACAT TATAGAAAAA GAAGGAGAGG TTATTTTCTT


 


GCATTTATTC ATGggggatc aaagctgatc tATAACTTCG


 


TATAGCATAC ATTATACGAA GTTATggtac actATTTAAA


 


TgcgCTCCAC TTGGCTCGGG GGGATATAGC TCAGTTGGTA


 


GAGCTCCGCT CTTGCAATTG GGTCGTTGCG ATTACGGGTT


 


GGATGTCTAA TTGTCCAGGC GGTAATGATA GTATCTTGTA


 


CCTGAACCGG TGGCTCACTT TTTCTAAGTA ATGGGGAAGA


 


GGACCGAAAC GTGCCACTGA AAGACTCTAC TGAGACAAAG


 


ATGGGCTGTC AAGAACGTAG AGGAGGTAGG ATGGGCAGTT


 


GGTCAGATCT AGTATGGATC GTACATGGAC GGTAGTTGGA


 


GTCGGCGGCT CTCCCAGGGT TCCCTCATCT GAGATCTCTG


 


GGGAAGAGGA TCAAGTTGGC CCTTGCGAAC AGCTTGATGC


 


ACTATCTCCC TTCAACCCTT TGAGCGAAAT GCGGCAAAAG


 


AAAAGGAAGG AAAATCCATG GACCGACCCC ATCATCTCCA


 


CCCCGTAGGA ACTACGAGAT CACCCCAAGG ACGCCTTCGG


 


CATCCAGGGG TCACGGACCG ACCATAGAAC CCTGTTCAAT


 


AAGTGGAACG CATTAGCTGT CCGCTCTCAG GTTGGGCAGT


 


CAGGGTCGGA GAAGGGCAAT GACTCATTCT TAGTTAGAAT


 


GGGATTCCAA CTCAGCACCT TTTGAGTGAG ATTTTGAGAA


 


GAGTTGCTCT TTGGAGAGCA CAGTACGATG AAAGTTGTAA


 


GCTGTGTTCG GGGGGGAGTT ATTGTCTATC GTTGGCCTCT


 


ATGGTAGAAT CAGTCGGGGG ACCTGAGAGG CGGTGGTTTA


 


CCCTGCGGCG GATGTCAGCG GTTCGAGTCC GCTTATCTCC


 


AACTCGTGAA CTTAGCCGAT ACAAAGCTCT GGCGTAATAG


 


CGAAGAGGCC CGCACCGATC GCCCTTCCCA ACAGTTGCGC


 


AGCCTGAATG GCGAATGGCG CCTGATGCGG TATTTTCTCC


 


TTACGCATCT GTGCGGTATT TCACACCGCA TACGTCAAAG


 


CAACCATAGT ACGCGCCCTG TAGCGGCGCA TTAAGCGCGG


 


CGGGTGTGGT GGTTACGCGC AGCGTGACCG CTACACTTGC


 


CAGCGCCCTA GCGCCCGCTC CTTTCGCTTT CTTCCCTTCC


 


TTTCTCGCCA CGTTCGCCGG CTTTCCCCGT CAAGCTCTAA


 


ATCCGGGGCT CCCTTTAGGG TTCCGATTTA GTGCTTTACG


 


GCACCTCGAC CCCAAAAAAC TTGATTTGGG TGATGGTTCA


 


CGTAGTGGGC CATCGCCCTG ATAGACGGTT TTTCGCCCTT


 


TGACGTTGGA GTCCACGTTC TTTAATAGTG GACTCTTGTT


 


CCAAACTGGA ACAACACTCA ACCCTATCTC GGGCTATTCT


 


TTTGATTTAT AAGGGATTTT GCCGATTTCG GCCTATTGGT


 


TAAAAAATGA GCTGATTTAA CAAAAATTTA ACGCGAATTT


 


TAACAAAATA TTAACGTTTA CAATTTTATG GTGCACTCTC


 


AGTACAATCT GCTCTGATGC CGCATAGTTA AGCCAGCCCC


 


GACACCCGCC AACACCCGCT GACGCGCCCT GACGGGCTTG


 


TCTGCTCCCG GCATCCGCTT ACAGACAAGC TGTGACCGTC


 


TCCGGGAGCT GCATGTGTCA GAGGTTTTCA CCGTCATCAC


 


CGAAACGCGC GAGACGAAAG GGCCTCGTGA TACGCCTATT


 


TTTATAGGTT AATGTCATGA TAATAATGGT TTCTTAGACG


 


TCAGGTGGCA CTTTTCGGGG AAATGTGCGC GGAACCCCTA


 


TTTGTTTATT TTTCTAAATA CATTCAAATA TGTATCCGCT


 


CATGAGACAA TAACCCTGAT AAATGCTTCA ATAATATTGA


 


AAAAGGAAGA GTATGAGTAT TCAACATTTC CGTGTCGCCC


 


TTATTCCCTT TTTTGCGGCA TTTTGCCTTC CTGTTTTTGC


 


TCACCCAGAA ACGCTGGTGA AAGTAAAAGA TGCTGAAGAT


 


CAGTTGGGTG CACGAGTGGG TTACATCGAA CTGGATCTCA


 


ACAGCGGTAA GATCCTTGAG AGTTTTCGCC CCGAAGAACG


 


TTTTCCAATG ATGAGCACTT TTAAAGTTCT GCTATGTGGC


 


GCGGTATTAT CCCGTATTGA CGCCGGGCAA GAGCAACTCG


 


GTCGCCGCAT ACACTATTCT CAGAATGACT TGGTTGAGTA


 


TTCACCAGTC ACAGAAAAGC ATCTTACGGA TGGCATGACA


 


GTAAGAGAAT TATGCAGTGC TGCCATAACC ATGAGTGATA


 


ACACTGCGGC CAACTTACTT CTGACAACGA TCGGAGGACC


 


GAAGGAGCTA ACCGCTTTTT TGCACAACAT GGGGGATCAT


 


GTAACTCGCC TTGATCGTTG GGAACCGGAG CTGAATGAAG


 


CCATACCAAA CGACGAGCGT GACACCACGA TGCCTGTAGC


 


AATGGCAACA ACGTTGCGCA AACTATTAAC TGGCGAACTA


 


CTTACTCTAG CTTCCCGGCA ACAATTAATA GACTGGATGG


 


AGGCGGATAA AGTTGCAGGA CCACTTCTGC GCTCGGCCCT


 


TCCGGCTGGC TGGTTTATTG CTGATAAATC TGGAGCCGGT


 


GAGCGTGGGT CTCGCGGTAT CATTGCAGCA CTGGGGCCAG


 


ATGGTAAGCC CTCCCGTATC GTAGTTATCT ACACGACGGG


 


GAGTCAGGCA ACTATGGATG AACGAAATAG ACAGATCGCT


 


GAGATAGGTG CCTCACTGAT TAAGCATTGG TAACTGTCAG


 


ACCAAGTTTA CTCATATATA CTTTAGATTG ATTTAAAACT


 


TCATTTTTAA TTTAAAAGGA TCTAGGTGAA GATCCTTTTT


 


GATAATCTCA TGACCAAAAT CCCTTAACGT GAGTTTTCGT


 


TCCACTGAGC GTCAGACCCC GTAGAAAAGA TCAAAGGATC


 


TTCTTGAGAT CCTTTTTTTC TGCGCGTAAT CTGCTGCTTG


 


CAAACAAAAA AACCACCGCT ACCAGCGGTG GTTTGTTTGC


 


CGGATCAAGA GCTACCAACT CTTTTTCCGA AGGTAACTGG


 


CTTCAGCAGA GCGCAGATAC CAAATACTGT CCTTCTAGTG


 


TAGCCGTAGT TAGGCCACCA CTTCAAGAAC TCTGTAGCAC


 


CGCCTACATA CCTCGCTCTG CTAATCCTGT TACCAGTGGC


 


TGCTGCCAGT GGCGATAAGT CGTGTCTTAC CGGGTTGGAC


 


TCAAGACGAT AGTTACCGGA TAAGGCGCAG CGGTCGGGCT


 


GAACGGGGGG TTCGTGCACA CAGCCCAGCT TGGAGCGAAC


 


GACCTACACC GAACTGAGAT ACCTACAGCG TGAGCTATGA


 


GAAAGCGCCA CGCTTCCCGA AGGGAGAAAG GCGGACAGGT


 


ATCCGGTAAG CGGCAGGGTC GGAACAGGAG AGCGCACGAG


 


GGAGCTTCCA GGGGGAAACG CCTGGTATCT TTATAGTCCT


 


GTCGGGTTTC GCCACCTCTG ACTTGAGCGT CGATTTTTGT


 


GATGCTCGTC AGGGGGGCGG AGCCTATGGA AAAACGCCAG


 


CAACGCGGCC TTTTTACGGT TCCTGGCCTT TTGCTGGCCT


 


TTTGCTCACA TGTTCTTTCC TGCGTTATCC CCTGATTCTG


 


TGGATAACCG TATTACCGCC TTTGAGTGAG CTGATACCGC


 


TCGCCGCAGC CGAACGACCG AGCGCAGCGA GTCAGTGAGC


 


GAGGAAGCGG AAGAGCGCCC AATACGCAAA CCGCCTCTCC


 


CCGCGCGTTG GCCGATTCAT TAATGCAG.


 


SEQ ID NO: 2


EcoRI-XbaI fragment in plasmid cry9Aa2pSKC84.seq


2277 nucleotides


  gaattccg tcgaggtcAA CCCAAATAAT GTTTTAAAAT


 


TTTAAAAATA ATGTAGGAGG AAAAATTATG AATCAAAATA


 


AACACGGAAT TATTGGCGCT TCCAATTGTG GTTGTGCATC


 


TGATGATGTT GCGAAATATC CTTTAGCCAA CAATCCATAT


 


TCATCTGCTT TAAATTTAAA TTCTTGTCAA AATAGTAGTA


 


TTCTCAACTG GATTAACATA ATAGGCGATG CAGCAAAAGA


 


AGCAGTATCT ATTGGGACAA CCATAGTCTC TCTTATCACA


 


GCACCTTCTC TTACTGGATT AATTTCAATA GTATATGACC


 


TTATAGGTAA AGTACTAGGA GGTAGTAGTG GACAATCCAT


 


ATCAGATTTG TCTATATGTG ACTTATTATC TATTATTGAT


 


TTACGGGTAA GTCAGAGTGT TTTAAATGAT GGGATTGCAG


 


ATTTTAATGG TTCTGTACTC TTATACAGGA ACTATTTAGA


 


GGCTCTGGAT AGCTGGAATA AGAATCCTAA TTCTGCTTCT


 


GCTGAAGAAC TCCGTACTCG TTTTAGAATC GCCGACTCAG


 


AATTTGATAG AATTTTAACC CGAGGGTCTT TAACGAATGG


 


TGGCTCGTTA GCTAGACAAA ATGCCCAAAT ATTATTATTA


 


CCTTCTTTTG CGAGCGCTGC ATTTTTCCAT TTATTACTAC


 


TAAGGGATGC TACTAGATAT GGCACTAATT GGGGGCTATA


 


CAATGCTACA CCTTTTATAA ATTATCAATC AAAACTAGTA


 


GAGCTTATTG AACTATATAC TGATTATTGC GTACATTGGT


 


ATAATCGAGG TTTCAACGAA CTAAGACAAC GAGGCACTAG


 


TGCTACAGCT TGGTTAGAAT TTCATAGATA TCGTAGAGAG


 


ATGACATTGA TGGTATTAGA TATAGTAGCA TCATTTTCAA


 


GTCTTGATAT TACTAATTAC CCAATAGAAA CAGATTTTCA


 


GTTGAGTAGG GTCATTTATA CAGATCCAAT TGGTTTTGTA


 


CATCGTAGTA GTCTTAGGGG AGAAAGTTGG TTTAGCTTTG


 


TTAATAGAGC TAATTTCTCA GATTTAGAAA ATGCAATACC


 


TAATCCTAGA CCGTCTTGGT TTTTAAATAA TATGATTATA


 


TCTACTGGTT CACTTACATT GCCGGTTAGC CCAAGTACTG


 


ATAGAGCGAG GGTATGGTAT GGAAGTCGAG ATCGAATTTC


 


CCCTGCTAAT TCACAATTTA TTACTGAACT AATCTCTGGA


 


CAACATACGA CTGCTACACA AACTATTTTA GGGCGAAATA


 


TATTTAGAGT AGATTCTCAA GCTTGTAATT TAAATGATAC


 


CACATATGGA GTGAATAGGG CGGTATTTTA TCATGATGCG


 


AGTGAAGGTT CTCAAAGATC CGTGTACGAG GGGTATATTC


 


GAACAACTGG GATAGATAAC CCTAGAGTTC AAAATATTAA


 


CACTTATTTA CCTGGAGAAA ATTCAGATAT CCCAACTCCA


 


GAAGACTATA CTCATATATT AAGCACAACA ATAAATTTAA


 


CAGGAGGACT TAGACAAGTA GCATCTAATC GCCGTTCATC


 


TTTAGTAATG TATGGTTGGA CACATAAAAG TCTGGCTCGT


 


AACAATACCA TTAATCCAGA TAGAATTACA CAGATACCAT


 


TGACGAAGGT TGATACCCGA GGCACAGGTG TTTCTTATGT


 


GAATGATCCA GGATTTATAG GAGGAGCTCT ACTTCAAAGG


 


ACTGACCATG GTTCGCTTGG AGTATTGAGG GTCCAATTTC


 


CACTTCACTT AAGACAACAA TATCGTATTA GAGTCCGTTA


 


TGCTTCTACA ACAAATATTC GATTGAGTGT GAATGGCAGT


 


TTCGGTACTA TTTCTCAAAA TCTCCCTAGT ACAATGAGAT


 


TAGGAGAGGA TTTAAGATAC GGATCTTTTG CTATAAGAGA


 


GTTTAATACT TCTATTAGAC CCACTGCAAG TCCGGACCAA


 


ATTCGATTGA CAATAGAACC ATCTTTTATT AGACAAGAGG


 


TCTATGTAGA TAGAATTGAG TTCATTCCAG TTAATCCGAC


 


GCGAGAGGCG AAAGAGGATC TAGAAGCAGC AAAAAAAGCG


 


GTGGCGAGCT TGTTTACACG CACAAGGGAC GGATTACAAG


 


TAAATGTGAA AGATTATCAA GTCGATCAAG CGGCAAATTT


 


AGTGTCATGC TTATCAGATG AACAATATGG GTATGACAAA


 


AAGATGTTAT TGGAAGCGGT ACGTGCGGCA AAACGACTTA


 


GCCGAGAACG CAACTTACTT CAGGATCCAt aattctaga


 


SEQ ID NO: 3


EcoRI-XbaI fragment in plasid pSKC85, with c-myc


tagged cry9Aa2 gene. 2307 nucleotides


  gaattccg tcgaggtcAA CCCAAATAAT GTTTTAAAAT


 


TTTAAAAATA ATGTAGGAGG AAAAATTATG AATCAAAATA


 


AACACGGAAT TATTGGCGCT TCCAATTGTG GTTGTGCATC


 


TGATGATGTT GCGAAATATC CTTTAGCCAA CAATCCATAT


 


TCATCTGCTT TAAATTTAAA TTCTTGTCAA AATAGTAGTA


 


TTCTCAACTG GATTAACATA ATAGGCGATG CAGCAAAAGA


 


AGCAGTATCT ATTGGGACAA CCATAGTCTC TCTTATCACA


 


GCACCTTCTC TTACTGGATT AATTTCAATA GTATATGACC


 


TTATAGGTAA AGTACTAGGA GGTAGTAGTG GACAATCCAT


 


ATCAGATTTG TCTATATGTG ACTTATTATC TATTATTGAT


 


TTACGGGTAA GTCAGAGTGT TTTAAATGAT GGGATTGCAG


 


ATTTTAATGG TTCTGTACTC TTATACAGGA ACTATTTAGA


 


GGCTCTGGAT AGCTGGAATA AGAATCCTAA TTCTGCTTCT


 


GCTGAAGAAC TCCGTACTCG TTTTAGAATC GCCGACTCAG


 


AATTTGATAG AATTTTAACC CGAGGGTCTT TAACGAATGG


 


TGGCTCGTTA GCTAGACAAA ATGCCCAAAT ATTATTATTA


 


CCTTCTTTTG CGAGCGCTGC ATTTTTCCAT TTATTACTAC


 


TAAGGGATGC TACTAGATAT GGCACTAATT GGGGGCTATA


 


CAATGCTACA CCTTTTATAA ATTATCAATC AAAACTAGTA


 


GAGCTTATTG AACTATATAC TGATTATTGC GTACATTGGT


 


ATAATCGAGG TTTCAACGAA CTAAGACAAC GAGGCACTAG


 


TGCTACAGCT TGGTTAGAAT TTCATAGATA TCGTAGAGAG


 


ATGACATTGA TGGTATTAGA TATAGTAGCA TCATTTTCAA


 


GTCTTGATAT TACTAATTAC CCAATAGAAA CAGATTTTCA


 


GTTGAGTAGG GTCATTTATA CAGATCCAAT TGGTTTTGTA


 


CATCGTAGTA GTCTTAGGGG AGAAAGTTGG TTTAGCTTTG


 


TTAATAGAGC TAATTTCTCA GATTTAGAAA ATGCAATACC


 


TAATCCTAGA CCGTCTTGGT TTTTAAATAA TATGATTATA


 


TCTACTGGTT CACTTACATT GCCGGTTAGC CCAAGTACTG


 


ATAGAGCGAG GGTATGGTAT GGAAGTCGAG ATCGAATTTC


 


CCCTGCTAAT TCACAATTTA TTACTGAACT AATCTCTGGA


 


CAACATACGA CTGCTACACA AACTATTTTA GGGCGAAATA


 


TATTTAGAGT AGATTCTCAA GCTTGTAATT TAAATGATAC


 


CACATATGGA GTGAATAGGG CGGTATTTTA TCATGATGCG


 


AGTGAAGGTT CTCAAAGATC CGTGTACGAG GGGTATATTC


 


GAACAACTGG GATAGATAAC CCTAGAGTTC AAAATATTAA


 


CACTTATTTA CCTGGAGAAA ATTCAGATAT CCCAACTCCA


 


GAAGACTATA CTCATATATT AAGCACAACA ATAAATTTAA


 


CAGGAGGACT TAGACAAGTA GCATCTAATC GCCGTTCATC


 


TTTAGTAATG TATGGTTGGA CACATAAAAG TCTGGCTCGT


 


AACAATACCA TTAATCCAGA TAGAATTACA CAGATACCAT


 


TGACGAAGGT TGATACCCGA GGCACAGGTG TTTCTTATGT


 


GAATGATCCA GGATTTATAG GAGGAGCTCT ACTTCAAAGG


 


ACTGACCATG GTTCGCTTGG AGTATTGAGG GTCCAATTTC


 


CACTTCACTT AAGACAACAA TATCGTATTA GAGTCCGTTA


 


TGCTTCTACA ACAAATATTC GATTGAGTGT GAATGGCAGT


 


TTCGGTACTA TTTCTCAAAA TCTCCCTAGT ACAATGAGAT


 


TAGGAGAGGA TTTAAGATAC GGATCTTTTG CTATAAGAGA


 


GTTTAATACT TCTATTAGAC CCACTGCAAG TCCGGACCAA


 


ATTCGATTGA CAATAGAACC ATCTTTTATT AGACAAGAGG


 


TCTATGTAGA TAGAATTGAG TTCATTCCAG TTAATCCGAC


 


GCGAGAGGCG AAAGAGGATC TAGAAGCAGC AAAAAAAGCG


 


GTGGCGAGCT TGTTTACACG CACAAGGGAC GGATTACAAG


 


TAAATGTGAA AGATTATCAA GTCGATCAAG CGGCAAATTT


 


AGTGTCATGC TTATCAGATG AACAATATGG GTATGACAAA


 


AAGATGTTAT TGGAAGCGGT ACGTGCGGCA AAACGACTTA


 


GCCGAGAACG CAACTTACTT CAGGATCCAg aacaaaaact


 


catttctgaa gaagacttgt gattctaga


 


SEQ ID NO: 4


DNA sequence encoding the processed 5′-UTR in


plasmids pSKC84 and pSKC85 including the ATG


translation initiation codon. The 49 Bt nucleo-


tides upstream of the ATG are in bold.


GCTGcATTTAAATGGCGCGCC gaattccg tcgaggtc AA


 


CCCAAATAAT GTTTTAAAAT TTTAAAAATA ATGTAGGAGG


 


AAAAATT ATG









While certain preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made to the invention without departing from the scope and spirit thereof as set forth in the following claims.

## Claims

1. A nucleic acid construct for high levels of translation for increasing heterologous protein production in a plastid of a higher plant, said construct comprising in the 5′ to 3′ direction,
a) a 5′ nucleic acid sequence comprising a 49 nucleotide segment of the native cry9Aa2 5′-UTR of SEQ ID NO:4;
b) a nucleic acid sequence encoding at least one heterologous protein or polypeptide of interest; and
c) a transcription termination region, said construct optionally further comprising a nucleic acid encoding a selectable marker,
wherein a), b) and c), and said selectable marker encoding nucleic acid, if present, are flanked by homologous nucleic acid sequences obtained from the genome of said plastid, said homologous nucleic acid sequences directing homologous recombination of said construct into said plastid genome, such that said heterologous protein or polypeptide is produced.

2. The construct of claim 1, wherein said heterologous protein is selected from the group consisting of at least one of antibodies, hormones, interferons, bacterial toxins, viral proteins, cytokines, proinsulin, antimicrobial peptides, insecticidal proteins, and enzymes suitable for the production of polyhydroxybutyrate (polyhydroxyalkanoate) polymers.

3. The construct of claim 1 comprising said selectable marker encoding nucleic acid, wherein expression of said marker confers resistance to an agent selected from the group consisting of spectinomycin, streptomycin, kanamycin, hygromycin, glyphosate, bromoxynil, phosphinothricine and sulfonylurea.

4. A plant cell comprising the construct of claim 1.

5. A plant cell plastid comprising the construct of claim 1.

6. A plant, plant seed, plant cell or progeny thereof comprising the construct of claim 2.

7. A method for producing a plant cell which expresses a heterologous protein of interest, said method comprising introducing the construct as claimed in claim 1 into said plant cell.

8. A plant regenerated from a plant cell produced by the method of claim 7.

9. A plant, plant seed, plant cell or progeny thereof comprising the plant cell of claim 4.

10. The construct of claim 1, wherein said at least one heterologous protein is selected from the group consisting of antibodies, hormones, interferons, bacterial toxins, viral proteins, cytokines, proinsulin, antimicrobial peptides, insecticidal proteins, and enzymes suitable for the production of polyhydroxybutyrate (polyhydroxyalkanoate) polymers
and said selectable marker encoding nucleic acid is present and wherein expression of said marker confers resistance to an agent selected from the group consisting of spectinomycin, streptomycin, kanamycin, hygromycin, glyphosate, bromoxynil, phosphinothricine and sulfonylurea.

11. A plant, plant seed, plant cell or progeny thereof comprising the construct of claim 10.

12. A vector comprising the construct of claim 1, wherein the vector targets insertion of said construct into an intergenic region of the plastid genome and expression of said construct lacks a promoter and expression of said protein occurs via read-through transcription from an endogenous upstream promoter.

13. The vector of claim 12, wherein said intergenic is trnI-trnA region and expression of said construct occurs via read through transcription of the plastid rRNA operon.

14. The construct of claim 1, comprising a promoter element operably linked 5′ to the nucleotide segment of a)