COMPOSITIONS AND METHODS FOR INCREASING TRANSGENE EXPRESSION IN THE PLASTIDS OF HIGHER PLANTS

By Pal Maliga

This application claims priority to US Provisional Application 60/792,853 filed April 18, 2006, the entire contents of which are 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 cry I class B.t. genes. Transgenic potato lines with variable level of PTM resistance have been obtained by expressing the native cry IAa (Chan et al., 1996), crylAb (Jansens et al., 1995) and cry IAc (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 crylAc9 (Beuning et al., 2001; Davidson et al., 2002), cry Hal (Mohammed et al., 2000; Douches et al., 2002), and a hybrid Bt toxin (SNl 9) gene consisting of domain I and III of cry IBa and domain II ofcrylla (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 (LC ₅₀ 80 ng/ml) (Gleave et al., 1992). The amino acid sequence of this new B.t. protein was significantly different from those belonging to Cryl 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 cry IAc (McBride et al., 1995), cry2Aa2 (Kota et al., 1999; De Cosa et al., 2001) and cryllaδ (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

Figure 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; rrnl6, trnl 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 EcoKL-Hindlll 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. BstEll digested total cellular DNA was

probed with the 2.1-kb EcoRI-Hmdlll ptDNA fragment to detect the wild type (4.6 kb) and transgenic (5.9 kb and 2.2 kb) BstEU fragments.

Figure 2. Testing mRNA accumulation by probing total cellular RNA with (A) cry9Aa2 (\ .3-kb Swal fragment), (B) aadA (0.8-kb Ncol-Xbal fragment) and (C) cytoplasmic 25S rRNA (loading control) probes.

Figure 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.

Figure 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. AU 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 trnl-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 US Patents 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 iner-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 trnVtrnh intergenic region in the plastid ribosomal RNA operon (GenBank Accesion Number DQ489715, SEQ ID NO: 1). Vector pPRV312L is a pUCl 18 plasmid derivative in which the Pvuϊl fragment was replaced with the Smal-Hindϊll 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 Pvuϊl-Sacl (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 oipsbA gene (Shinozaki et al., 1986). The aadA gene is flanked by the Pl phage loxP sites to facilitate its excision by the CRE site-specific recombinase (Corneille et al., 2001; Lutz et al., 2006).

Plasmid pNZAlO carrying the cry9Aa2 gene (GenBank Accession number X58534) was obtained from the Bacillus Genetic Stock Center, The Ohio State University, Columbus, Ohio. First the HinclI/BamHl fragment (2.2 Kbp) encoding 49 nucleotides of the leader sequence and the N-terminal half of the cry9Aa2 gene was excised from plasmid pNZAlO and sub-cloned into a pBluescriptKS (Stratagene, La Jolla, CA) plasmid. The C-terminally truncated cry9Aa2 gene was converted into an EcoRI-Xbal fragment for expression in pPRV312L. ThcXhol site upstream of the Hindi was converted into an EcoRI site by blunting and linker ligation and an in- frame stop codon was introduced in a BamHI-Xbal linker (5' -GGATCC Ataattctaga- 3'). The modified cry9Aa2 gene was excised as an EcoKL-Xbal fragment (SEQ ID NO: 2) and cloned in a plasmid pPRV312L derivative, which carried the 3'UTR of the rbcL gene (TrbcL; Xbal-Hinάlll 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) (Kolodziej &Young, 1991) introduced in a BamHI-Xbal fragment (5'-

GGATCCgaacaaaaactcatttctgaagaagacttgtgattctaga-3') with the stop codon. The DNA sequence of the EcoRI-Xbal 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, CA). Transforming DNA was introduced into leaf chloroplasts on the surface of tungsten particles (1 μm) using the Du Pont

PDSlOOOHe 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 BstEll restriction enzyme. Double- stranded DNA probes were prepared by random-primed P-labeling using the Ready- To-Go DNA Labeling Beads (Amershem Pharmacia Biotech, Piscataway, NJ). The probe was the trnl-trnA plastid targeting region, encoded in an EcøRI-HmdIII 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 ³² P-labeling (see above). The cry9Aa2 and aadA probes were prepared using Swal and Ncol-Xbal 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 ⁰ C. The insoluble material was then removed by centrifugation. Soluble protein concentrations were determined by the Bradford Protein Assay Reagent kit (Bio-Rad, Hercules, CA); membrane proteins were quantified with the bicinchoninic acid (BCA) method (Pierce, Rockford, IL). The protein in the Comassie Blue stained soluble extracts was quantified with Alpha Innotech (San Leandro, CA) 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, CA).

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 ⁰ 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 trnl-trnA intergenic region located between the rrnlό and rrn23 genes in the plastid rRNA operon. The pPRV312L vector carries a selectable spectinomcyin resistance (aadA) gene flanked by /ox-sites for marker gene excision, and a multiple cloning site for passenger genes (Figure IA). 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) (Figure IA). 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 (Figure IB). 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 ofcry9Aa2 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 (Figure 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 trnl and the 3 '-end by processing within the TrbcL or at the /øxP-site. In RNA samples from young leaves additional, partially processed mRNA species are visible, which are processing intermediates of the rrn operon transcript (Figure IA). Degradation of cry9Aα2 mRNA is apparent in each of the samples.

The selective marker ααdA is transcribed from two promoters: its own promoter (PrrnLatpB+DS) and the native rrn operon promoter upstream of rrnl 6. Probing with ααdA revealed two ~l.l-kb monocistronic messages, which did not separate on the blot shown in Figure 2. These transcripts derive from the ααdA 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 (Figure 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 (Figure 3A). Immunoblot analysis showed that the Cry9Aa2 protein concentration in the membrane fraction was higher, ~ 20% (Figure 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 (Figure 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 (Figure 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 pPR V312L plastid vector

The new vector pPRV312L has a multiple cloning site and /øxP-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 pPRVl 1OL (GenBank Accession No.

DQ21 1347) that targets insertions in the trnV-rpsl2 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 thepsbA 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, 2001a; Kuroda &Maliga, 2001b); for review see (Maliga, 2003). Thus, the 49 nucleotide Bj. cry9aA2 5'-UTR is a leader that promotes high-level protein expression in chloroplasts. The 49 Bt nucleotide sequence is: 5 '- AACCCAAATAATGTTTTAAAATTTTAAAAATAA TGTAGGAGGAAAAATT-3'. 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 BJ. 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 rrnlό, trnl, 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-modifϊed 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% cry IAc (McBride et al., 1995), 3% (Kota et al., 1999) and 45.3% (De Cosa et al., 2001) cry2Aa2 and 3% cryllaδ (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 trnl/trnA intergenic region; in pUCllδ vector derivative (circular DNA)

pPRV312L.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 AΆAACATTTC 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 ATCGGGGGCT 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 GAGACGAΆAG

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-Xbal 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-Xbal 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 nucleotides 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.