US20120174253A1

US20120174253A1 - Generation of high polyhydroxybutrate producing oilseeds

Info

Publication number: US20120174253A1
Application number: US13/395,616
Authority: US
Inventors: Nii Patterson; Jihong Tang; Edgar Benjamin Cahoon; Jan G. Jaworski; Wenyu Yang; Oliver P. Peoples; Kristi D. Snell
Original assignee: Individual
Current assignee: Donald Danforth Plant Science Center; Yield10 Bioscience Inc
Priority date: 2009-09-15
Filing date: 2010-09-15
Publication date: 2012-07-05
Also published as: BR112012005592A2; AU2010295637A1; CA2773703A1; BR112012005591A2; US20120180162A1; US9181559B2; WO2011034946A9; WO2011034946A1; AU2010295637B2; EP2478105A1; CA2773707A1; AU2010295638A1; WO2011034945A1; EP2477477A1

Abstract

Transgenic oilseed plants, plant material, plant cells, and genetic constructs for synthesis of polyhydroxyalkanoates (“PHA”) are provided. In a preferred embodiment, the transgenic oilseed plants synthesize (poly)3-hydroxybutyrate (“PHB”) in the seed. Genes utilized include phaA, phaB, phaC, all of which are known in the art. The genes can be introduced in the plant, plant tissue, or plant cell using conventional plant molecular biology techniques.

Description

FIELD OF THE INVENTION

The invention is in the field of polymer production in transgenic plants. Methods for generating industrial oilseeds producing high levels of polyhydroxybutyrate (PHB) and industrial oilseeds producing high levels of PHB are described.

BACKGROUND OF THE INVENTION

Production of polyhydroxyalkanoates (PHAs), a family of naturally occurring renewable and biodegradable plastics, in crops has the potential of providing a renewable source of polymers, chemical intermediates and bio-energy from one crop if plant residues remaining after polymer isolation are converted to liquid fuels and/or energy. PHAs can provide an additional revenue stream that would make bioenergy crops more economically viable.
PHAs are a natural component of numerous organisms in multiple ecosystems and accumulate in a wide range of bacteria as a granular storage material when the microbes are faced with an unfavorable growth environment, such as a limitation in an essential nutrient (Madison et al., Microbiol. Mol. Biol. Rev., 1999, 63, 21-53; Suriyamongkol et al., Biotechnol Adv, 2007, 25, 148-175). The monomer unit composition of these polymers is largely dictated by available carbon source as well as the native biochemical pathways present in the organism. Today PHAs are produced industrially from renewable resources in bacterial fermentations providing an alternative to plastics derived from fossil fuels. PHAs possess properties enabling their use in a variety of applications currently served by petroleum-based plastics and are capable of matching or exceeding the performance characteristics of fossil fuel derived plastics with a broad spectrum of properties that can be obtained by varying the monomer composition of homo- and co-polymers, or by manipulating properties such as molecular weight (Sudesh et al., Prog. Polym. Sci., 2000, 25, 1503-1555; Sudesh et al., CLEAN—Soil, Air, Water, 2008, 36, 433-442).
Industrial production of PHAs in crop plants would provide a low cost, renewable source of plastics. Production of PHAs in plants has been an as yet unsolved goal for plant scientists and has previously been demonstrated in a number of crops unsuitable for industrial production or in industrially useful crops at levels to low to be commercially attractive [for review, see (Suriyamongkol et al., Biotechnol Adv, 2007, 25, 148-175); (van Beilen et al., The Plant Journal, 2008, 54, 684-701) and references within] including maize (Poirier et al., 2002, Polyhydroxyalkanoate production in transgenic plants, in Biopolymers, Vol 3a, Steinbuchel, A. (ed), Wiley-VHC Verlag GmbH, pgs 401-435), sugarcane (Purnell et al., Plant Biotechnol. J., 2007, 5, 173-184), switchgrass (Somleva et al., Plant Biotechnol J, 2008, 6, 663-678), flax (Wrobel et al., J. Biotechnol., 2004, 107, 41-54; Wrobel-Kwiatkowsk et al., Biotechnol Prog, 2007, 23, 269-277), cotton (John et al., Proceedings of the National Academy of Sciences of the United States of America, 1996, 93, 12768-12773), alfalfa (Saruul et al., Crop Sci., 2002, 42, 919-927), tobacco (Arai et al., Plant Biotechnol., 2001, 18, 289-293; Bohmert et al., Plant Physiol., 2002, 128, 1282-1290; Lossl et al., Plant Cell Reports, 2003, 21, 891-899; Lössl et al., Plant Cell Physiol, 2005, 46, 1462-1471), potato (Bohmert et al., Plant Physiol., 2002, 128, 1282-1290), and oilseed rape (Valentin et al., Int. J. Biol. Macromol., 1999, 25, 303-306; Slater et al., Nat. Biotechnol., 1999, 17, 1011-1016). Most of the efforts to produce PHAs in plants have focused on production of the homopolymer P3HB or the copolymer poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P3HBV). While there have been some efforts to produce medium chain length PHAs in plants, these studies have yielded barely detectable levels of polymer (Romano et al., Planta, 2005, 220, 455-464; Mittendorf et al., Proceedings of the National Academy of Sciences of the United States of America, 1998, 95, 13397-13402; Poirier et al., Plant Physiol., 1999, 121, 1359-1366; Matsumoto, Journal of Polymers and the Environment, 2006, 14, 369-374; Wang et al., Chinese Science Bulletin, 2005, 50, 1113-1120).
To date, the highest levels of polymer have been obtained when the homopolymer poly-3-hydroxybutyrate (P3HB or PHB) is produced in plastids (Suriyamongkol et al., Biotechnol Adv, 2007, 25, 148-175; van Beilen et al., The Plant Journal, 2008, 54, 684-701; Bohmert et al., Molecular Biology and Biotechnology of Plant Organelles, 2004, 559-585). This is likely due to the high flux of acetyl-CoA, the precursor for PHB in these organelles during fatty acid biosynthesis (Bohmert et al, Molecular Biology and Biotechnology of Plant Organelles, 2004, 559-585). Expression of three genes encoding β-ketothiolase, acetoacetyl CoA reductase, and PHA synthase, allows the conversion of acetyl-CoA within the plastid to PHB. Previous work has reported producing levels of PHB in Brassica napus up to a maximum of 6.7% of seed weight, a level too low for commercial production

SUMMARY OF THE INVENTION

Transgenic oilseed plants, plant material, plant cells, and genetic constructs for synthesis of polyhydroxyalkanoates (“PHA”) are provided. In a preferred embodiment, the transgenic oilseed plants synthesize (poly)3-hydroxybutyrate (“PHB”) in the seed. Host plants, plant tissue, and plant material have been engineered to express genes encoding enzymes in the biosynthetic pathway for PHB production such that polymer precursors in the plastid are polymerized to polymer. Genes utilized include phaA, phaB, phaC, all of which are known in the art. The genes can be introduced in the plant, plant tissue, or plant cell using conventional plant molecular biology techniques.
It is an object of the invention to provide methods and compositions for producing transgenic oilseeds having commercially viable levels of polyhydroxyalkanoates in the seed, for example greater than 7%, 10%, 15%, or 19% polyhydroxyalkanoate or more of the total dry seed weight.
It is another object of the invention to provide oilseeds having increased levels of polyhydroxyalkanoate greater than 7%, 10%, 15%, or 19% polyhydroxyalkanoate or more of the total dry seed weight and having impaired germination relative to non-transgenic oilseeds.
Using a non-traditional screening method to identify transgenic lines than those used in all other reported studies, it has been discovered that very high levels of PHA, for example PHB can be produced in the oilseed but that oilseeds with high levels of PHA fail to germinate or germinate but produce impaired seedlings which do not survive to produce viable fertile plants. The failure to produce viable progeny explains why previous researchers failed to demonstrate that commercial levels of PHA can be produced in transgenic oilseeds. A preferred PHA produced in oilseeds is PHB.
In another embodiment the transgenes encoding PHA biosynthesis genes are expressed in a seed specific manner such that the PHA accumulates in the seed. In this embodiment the level of PHA accumulated is greater than 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% and 19% of the dry weight of the seed.
Methods and compositions for producing hybrid lines are also provided. Hybrid lines can be created by crossing a line containing one or more PHAs, for example PHB genes with a line containing the other gene(s) needed to complete the PHA biosynthetic pathway. Use of lines that possess cytoplasmic male sterility with the appropriate maintainer and restorer lines allows these hybrid lines to be produced efficiently.
In still another embodiment the oilseeds produced by the disclosed methods produce high levels of PHA and are impaired in their ability to germinate and survive to produce viable plants relative to oilseeds containing little or no PHA, for example less than 7% PHA of the dry weight of the seed. Germination can be impaired by 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% relative to oilseeds with less than 7% PHA. Impaired germination provides a built in mechanism for gene containment reducing the risk of unwanted growth of these oilseeds when a different crop is planted on the production fields.
Transgenic plants useful for the invention include dicots or monocots. Preferred host plants are oilseed plants, but are not limited to members of the Brassica family including B. napus, B. rapa, B. carinata and B. juncea. Additional preferred host plants include industrial oilseeds such as Camelina sativa, Crambe, jatropha, and castor. Other preferred host plants include Arabidopsis thaliana, Calendula, Cuphea, maize, soybean, cottonseed, sunflower, palm, coconut, safflower, peanut, mustards including Sinapis alba, and tobacco.
Other embodiments provide plant material and plant parts of the transgenic plants including seeds, flowers, stems, and leaves. The oilseeds can be used for the extraction of PHA biopolymer or as a source of PHA biopolymer based chemical intermediates. The residual parts of the seed can be used as meal for animal feed or steam and power generation and a source of vegetable oil for industrial oelochemicals or biofuel.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram describing a strategy for creating hybrid seeds using cytoplasmic male sterility.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

Unless otherwise indicated, the disclosure encompasses all conventional techniques of plant breeding, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition (2001); Current Protocols In Molecular Biology [(F. M. Ausubel, et al. eds., (1987)]; Plant Breeding Principles and Prospects (Plant Breeding, Vol 1) M. D. Hayward, N. O. Bosemark, I. Romagosa; Chapman & Hall, (1993.); Coligan, Dunn, Ploegh, Speicher and Wingfeld, eds. (1995) Current Protocols in Protein Science (John Wiley & Sons, Inc.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)].
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Lewin, Genes VII, published by Oxford University Press, 2000; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Wiley-Interscience., 1999; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology, a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; Ausubel et al. (1987) Current Protocols in Molecular Biology, Green Publishing; Sambrook and Russell. (2001) Molecular Cloning: A Laboratory Manual 3rd. edition.
A number of terms used herein are defined and clarified in the following section.
The term PHB refers to polyhydroxybutyrate and is used interchangeably with the term PHA which refers to polyhydroxyalkanoate.
The tend PHB also encompasses copolymers of hydroxybutyrate with other hydroxyacid monomers.
The term “PHA copolymer” refers to a polymer composed of at least two different hydroxyalkanoic acid monomers.
The term “PHA homopolymer” refers to a polymer that is composed of a single hydroxyalkanoic acid monomer.
As used herein, a “vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. The vectors can be expression vectors.
As used herein, an “expression vector” is a vector that includes one or more expression control sequences
As used herein, an “expression control sequence” is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence. Control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and the like. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
As used herein, “operably linked” means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest.
As used herein, “transformed” and “transfected” encompass the introduction of a nucleic acid into a cell by a number of techniques known in the art.
“Plasmids” are designated by a lower case “p” preceded and/or followed by capital letters and/or numbers.
As used herein the term “heterologous” means from another host. The other host can be the same or different species.
The term “cell” refers to a membrane-bound biological unit capable of replication or division.
The term “construct” refers to a recombinant genetic molecule including one or more isolated polynucleotide sequences.
Genetic constructs used for transgene expression in a host organism comprise in the 5′-3′ direction, a promoter sequence; a nucleic acid sequence encoding the desired transgene product; and a termination sequence. The open reading frame may be orientated in either a sense or anti-sense direction. The construct may also comprise selectable marker gene(s) and other regulatory elements for expression.
The term “plant” is used in it broadest sense. It includes, but is not limited to, any species of woody, ornamental or decorative, crop or cereal, fruit or vegetable plant, and photosynthetic green algae (e.g., Chlamydomonas reinhardtii). It also refers to a plurality of plant cells that are largely differentiated into a structure that is present at any stage of a plant's development. Such structures include, but are not limited to, a fruit, shoot, stem, leaf, flower petal, etc. The term “plant tissue” includes differentiated and undifferentiated tissues of plants including those present in roots, shoots, leaves, pollen, seeds and tumors, as well as cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.). Plant tissue may be in planta, in organ culture, tissue culture, or cell culture. The term “plant part” as used herein refers to a plant structure, a plant organ, or a plant tissue.
A non-naturally occurring plant refers to a plant that does not occur in nature without human intervention. Non-naturally occurring plants include transgenic plants and plants produced by non-transgenic means such as plant breeding.
The term “plant cell” refers to a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, a plant organ, or a whole plant.
The term “plant cell culture” refers to cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.
The term “plant material” refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.
A “plant organ” refers to a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.
“Plant tissue” refers to a group of plant cells organized into a structural and functional unit. Any tissue of a plant, whether in a plant or in culture, is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.
“Seed germination” refers to growth of an embryonic plant contained within a seed resulting in the formation and emergence of a seedling.
“Cotyledon” refers to the embryonic first leaves of a seedling.
“Early plantlet development” refers to growth of the cotyledon containing seedling to form a plantlet.

II. Transgenic Plants

Transgenic plants have been developed that produce increased levels of biopolymers such as polyhydroxyalkanoates (PHAs) in seeds. Methods and constructs for engineering plants for seed specific production of PHA, in particular PHB, are described. One embodiment provides transgenic plants for the direct, large scale production of PHAs in crop plants or in energy crops where a plant by-product, such as oil, can be used for production of energy. Proof of concept studies for polyhydroxybutyrate (PHB) synthesis in canola (Valentin et al., Int. J. Biol. Macromol., 1999, 25, 303-306; Houmiel et al., Planta, 1999, 209, 547-550; Slater et al., Nat. Biotechnol., 1999, 17, 1011-1016.) have been reported. There have been instances where high level PHB production in the chloroplasts of plants has led to decreases in total plant growth (Bohmert et al., Molecular Biology and Biotechnology of Plant Organelles, 2004, 559-585; Bohmert et al., Planta, 2000, 211, 841-845) for unidentified reasons. There have been several studies that have attempted to alleviate this problem by inducible expression of enzymes (Bohmert et al., Plant Physiol., 2002, 128, 1282-1290; Lössl et al., Plant Cell Physiol, 2005, 46, 1462-1471; Kourtz et al., Transgenic Res, 2007, 16, 759-769).
Transgenic oilseeds comprising at least about 8% dry weight PHA are provided. In one embodiment we provide transgenic oilseeds having at least 10% PHA dry weight and which are impaired in germination and plant survival.
A. Genetic Constructs for Transformation
Suitable genetic constructs include expression cassettes for enzymes for production of polyhydroxyalkanoates, in particular from the polyhydroxybutyrate biosynthetic pathway. In one embodiment, the construct contains operatively linked in the 5′ to 3′ direction, a seed specific promoter that directs transcription of a nucleic acid sequence in the nucleus; a nucleic acid sequence encoding one of the PHB biosynthetic enzymes; and a 3′ polyadenylation signal that increases levels of expression of transgenes. In one embodiment, enzymes for formation of polymer precursors are targeted to the plastid using appropriate plastid-targeting signals. In another embodiment, the PHA pathway is expressed directly from the plastid genome using appropriate plastidial promoters and regulatory sequences.
DNA constructs useful in the methods described herein include transformation vectors capable of introducing transgenes into plants. As used herein, “transgenic” refers to an organism in which a nucleic acid fragment containing a heterologous nucleotide sequence has been introduced. The transgenes in the transgenic organism are preferably stable and inheritable. The heterologous nucleic acid fragment may or may not be integrated into the host genome.
Several plant transformation vector options are available, including those described in “Gene Transfer to Plants” (Potrykus, et al., eds.) Springer-Verlag Berlin Heidelberg New York (1995); “Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins” (Owen, et al., eds.) John Wiley & Sons Ltd. England (1996); and “Methods in Plant Molecular Biology: A Laboratory Course Manual” (Maliga, et al. eds.) Cold Spring Laboratory Press, New York (1995). Plant transformation vectors generally include one or more coding sequences of interest under the transcriptional control of 5′ and 3′ regulatory sequences, including a promoter, a transcription termination and/or polyadenylation signal, and a selectable or screenable marker gene. For the expression of two or more polypeptides from a single transcript, additional RNA processing signals and ribozyme sequences can be engineered into the construct (U.S. Pat. No. 5,519,164). This approach has the advantage of locating multiple transgenes in a single locus, which is advantageous in subsequent plant breeding efforts.
Engineered minichromosomes can also be used to express one or more genes in plant cells. Cloned telomeric repeats introduced into cells may truncate the distal portion of a chromosome by the formation of a new telomere at the integration site. Using this method, a vector for gene transfer can be prepared by trimming off the arms of a natural plant chromosome and adding an insertion site for large inserts (Yu et al., Proc Natl Acad Sci USA, 2006, 103, 17331-6; Yu et al., Proc Natl Acad Sci USA, 2007, 104, 8924-9). The utility of engineered minichromosome platforms has been shown using Cre/lox and FRT/FLP site-specific recombination systems on a maize minichromosome where the ability to undergo recombination was demonstrated (Yu et al., Proc Natl Acad Sci USA, 2006, 103, 17331-6; Yu et al., Proc Natl Acad Sci USA, 2007, 104, 8924-9). Such technologies could be applied to minichromosomes, for example, to add genes to an engineered plant. Site specific recombination systems have also been demonstrated to be valuable tools for marker gene removal (Kerbach, S. et al., Theor Appl Genet, 2005, 111, 1608-1616), gene targeting (Chawla, R. et al., Plant Biotechnol J, 2006, 4, 209-218; Choi, S. et al., Nucleic Acids Res, 2000, 28, E19; Srivastava, V, & Ow, D W, Plant Mol Biol, 2001, 46, 561-566; Lyznik, L A, et al., Nucleic Acids Res, 1993, 21, 969-975), and gene conversion (Djukanovic, V, et al., Plant Biotechnol J, 2006, 4, 345-357).
An alternative approach to chromosome engineering in plants involves in vivo assembly of autonomous plant minichromosomes (Carlson et al., PLoS Genet, 2007, 3, 1965-74). Plant cells can be transformed with centromeric sequences and screened for plants that have assembled autonomous chromosomes de novo. Useful constructs combine a selectable marker gene with genomic DNA fragments containing centromeric satellite and retroelement sequences and/or other repeats.
Another approach is Engineered Trait Loci (“ETL”) technology (U.S. Pat. No. 6,077,697 to Hadlaczky et al.; US Patent Application 2006/0143732). This system targets DNA to a heterochromatic region of plant chromosomes, such as the pericentric heterochromatin, in the short arm of acrocentric chromosomes. Targeting sequences may include ribosomal DNA (rDNA) or lambda phage DNA. The pericentric rDNA region supports stable insertion, low recombination, and high levels of gene expression. This technology is also useful for stacking of multiple traits in a plant (US Patent Application 2006/0246586, 2010/0186117 and PCT WO 2010/037209).
Zinc-finger nucleases (ZFNs) are also useful in that they allow double strand DNA cleavage at specific sites in plant chromosomes such that targeted gene insertion or deletion can be performed (Shukla et al., Nature, 2009; Townsend et al., Nature, 2009).
For direct expression of transgenes from the plastid genome, a vector to transform the plant plastid chromosome by homologous recombination (as described in U.S. Pat. No. 5,545,818 to McBride et al.) is used in which case it is possible to take advantage of the prokaryotic nature of the plastid genome and insert a number of transgenes as an operon. WO 2010/061186 describes an alternative method for introducing genes into the plastid chromosome using an adapted endogenous cellular process for the transfer of RNAs from the cytoplasm to the plastid where they are incorporated by homologous recombination. This plastid transformation procedure is also suitable for practicing the disclosed compositions and methods.
A transgene may be constructed to encode a multifunctional enzyme through gene fusion techniques in which the coding sequences of different genes are fused with or without linker sequences to obtain a single gene encoding a single protein with the activities of the individual genes. Transgenes encoding a bifunctional protein containing thiolase and reductase activities (Kourtz, L., K. et al. (2005), Plant Biotechnol. 3: 435-447) and a trifunctional protein having each of the three enzyme activities required for PHB expression in plants (Mullaney and Rehm (2010), Journal of Biotechnology 147: 31-36) have been described. Such synthetic fusion gene/enzyme combinations can be further optimized using molecular evolution technologies.
A transgene may be constructed to encode a series of enzyme activities separated by intein sequences such that on expression, two or more enzyme activities are expressed from a single promoter as described by Snell in U.S. Pat. No. 7,026,526 to Metabolix, Inc.
1. Genes Involved in Polyhydroxyalkanoate Synthesis
In a preferred embodiment, the products of the transgenes are enzymes and other factors required for production of a biopolymer, such as a polyhydroxyalkanoate (PHA).
For PHA production, transgenes encode enzymes such as beta-ketothiolase, acetoacetyl-CoA reductase, PHB (“short chain”) synthase, PHA (“long chain”) synthase, threonine dehydratase, dehydratases such as 3-OH acyl ACP, isomerases such as Δ 3-cis, Δ 2-trans isomerase, propionyl-CoA synthetase, hydroxyacyl-CoA synthetase, hydroxyacyl-CoA transferase, R-3-hydroxyacyl-ACP:CoA transferase, thioesterase, fatty acid synthesis enzymes and fatty acid beta-oxidation enzymes. Useful genes are well known in the art, and are disclosed for example by Snell and Peoples Metab. Eng. 4: 29-40 (2002); Bohmert et. al. in Molecular Biology and Biotechnology of Plant Organelles. H. Daniell, C. D. Chase Eds., Kluwer Academic Publishers, Netherlands, 2004, pp. 559-585; (Suriyamongkol et al., Biotechnol Adv, 2007, 25, 148-175; van Beilen et al., The Plant Journal, 2008, 54, 684-701).
PHA Synthases
Examples of PHA synthases include a synthase with medium chain length substrate specificity, such as phaC1 from Pseudomonas oleovorans (WO 91/000917; Huisman, et al. J. Biol. Chem. 266, 2191-2198 (1991)) or Pseudomonas aeruginosa (Timm, A. & Steinbuchel, A. Eur. J. Biochem. 209: 15-30 (1992)), the synthase from Alcaligenes eutrophus with short chain length specificity (Peoples, O. P. & Sinskey, A. J. J. Biol. Chem. 264:15298-15303 (1989)), or a two subunit synthase such as the synthase from Thiocapsa pfennigii encoded by phaE and phaC (U.S. Pat. No. 6,011,144). Other useful PHA synthase genes have been isolated from, for example, Alcaligenes latus (Accession ALU47026), Burkholderia sp. (Accession AF153086), Aeromonas caviae (Fukui & Doi, J. Bacteriol. 179: 4821-30 (1997)), Acinetobacter sp. strain RA3849 (Accession L37761), Rhodospirillum rubrum (U.S. Pat. No. 5,849,894), Rhodococcus ruber (Pieper & Steinbuechel, FEMS Microbiol. Lett. 96(1): 73-80 (1992)), and Nocardia corallina (Hall et. al., Can. J. Microbiol. 44: 687-91 (1998)), Arthrospira sp. PCC 8005 (Accessions ZP_—07166315 and ZP_—07166316), Cyanothece sp. PCC 7425 (Accessions ACL46371 and ACL46370) and Synechocystis sp. PCC6803 (Accession BAA17430; Hein et al. (1998), Archives of Microbiology 170: 162-170).
PHA synthases with broad substrate specificity useful for producing copolymers of 3-hydroxybutyrate and longer chain length (from 6 to 14 carbon atoms) hydroxyacids have also been isolated from Pseudomonas sp. A33 (Appl. Microbiol. Biotechnol. 42: 901-909 (1995)) and Pseudomonas sp. 61-3 (Accession AB014757; Kato, et al. Appl. Microbiol. Biotechnol. 45: 363-370 (1996)).
A range of PHA synthase genes and genes encoding additional metabolic steps useful in PHA biosynthesis are described by Madison and Huisman. Microbiology and Molecular biology Reviews 63:21-53 (1999)) and Suriyamongkol et al. (Suriyamongkol et al., Biotechnol Adv, 2007, 25, 148-175).
Hydratase and Dehydrogenase
An alpha subunit of beta-oxidation multienzyme complex pertains to a multifunctional enzyme that minimally possesses hydratase and dehydrogenase activities. The subunit may also possess epimerase and A 3-cis, A 2-trans isomerase activities. Examples of alpha subunits of the beta-oxidation multienzyme complex are FadB from E. coli (DiRusso, C. C. J. Bacteriol. 1990, 172, 6459-6468), FaoA from Pseudomonas fragi (Sato, S., Hayashi, et al. J. Biochem. 1992, 111, 8-15), and the E. coli open reading frame 1714 that contains homology to multifunctional α subunits of the β-oxidation complex (Genbank Accession #1788682). A β subunit of the β-oxidation complex refers to a polypeptide capable of forming a multifunctional enzyme complex with its partner α subunit. The β subunit possesses thiolase activity. Examples of β subunits are FadA from E. coli (DiRusso, C. C. J. Bacteriol. 172: 6459-6468 (1990)), FaoB from Pseudomonas fragi (Sato, S., Hayashi, M., Imamura, S., Ozeki, Y., Kawaguchi, A. J. Biochem. 111: 8-15 (1992)), and the E. coli open reading frame f436 that contains homology to α subunits of the β-oxidation complex (Genbank Accession # AE000322; gene b2342).
Reductases
The transgene can encode a reductase. A reductase refers to an enzyme that can reduce β-ketoacyl CoAs to R-3-OH-acyl CoAs, such as the NADH dependent reductase from Chromatium vinosum (Liebergesell, M., & Steinbuchel, A. Eur. J. Biochem. 209: 135-150 (1992)), the NADPH dependent reductase from Alcaligenes eutrophus (Accession J04987, Peoples, O. P. & Sinskey, A. J. J. Biol. Chem. 264: 15293-15297 (1989))), the NADPH reductase from Zoogloea ramigera (Accession P23238; Peoples, O. P. & Sinskey, A. J. Molecular Microbiology 3: 349-357 (1989)) or the NADPH reductase from Bacillus megaterium (U.S. Pat. No. 6,835,820), Alcaligenes latus (Accession ALU47026), Rhizobium meliloti (Accession RMU17226), Paracoccus denitrificans (Accession D49362), Burkholderia sp. (Accession AF153086), Pseudomonas sp. strain 61-3 (Accession AB014757), Acinetobacter sp. strain RA3849 (Accession L37761), P. denitrificans, (Accession P50204), and Synechocystis sp. Strain PCC6803 (Taroncher-Oldenburg et al., (2000), Appl. Environ. Microbiol. 66: 4440-4448).
Thiolases
The transgene can encode a thiolase. A beta-ketothiolase refers to an enzyme that can catalyze the conversion of acetyl CoA and an acyl CoA to a β-ketoacyl CoA, a reaction that is reversible. An example of such thiolases are PhaA from Alcaligenes eutropus (Accession J04987, Peoples, O. P. & Sinskey, A. J. J. Biol. Chem. 264: 15293-15297 (1989)), BktB from Alcaligenes eutrophus (Slater et al. J Bacteriol. 180(8):1979-87 (1998)) and thiolases from the following Rhizobium meliloti (Accession RMU17226), Z. ramigera (Accession P07097), Paracoccus denitrificans (Accession D49362), Burkholderia sp. (Accession AF153086), Alcaligenes latus (Accession ALU47026), Allochromatium vinosum (Accession P45369), Thiocystis violacea (Accession P45363); Pseudomonas sp. strain 61-3 (Accession AB014757), Acinetobacter sp. strain RA3849 (Accession L37761) and Synechocystis sp. Strain PCC6803 (Taroncher-Oldenburg et al., (2000), Appl. Environ. Microbiol. 66: 4440-4448).
Oxidases
An acyl CoA oxidase refers to an enzyme capable of converting saturated acyl CoAs to Δ 2 unsaturated acyl CoAs. Examples of acyl CoA oxidases are POX1 from Saccharomyces cerevisiae (Dmochowska, et al. Gene, 1990, 88, 247-252) and ACX1 from Arabidopsis thaliana (Genbank Accession # AF057044).
Catalases
The transgene can also encode a catalase. A catalase refers to an enzyme capable of converting hydrogen peroxide to hydrogen and oxygen. Examples of catalases are KatB from Pseudomonas aeruginosa (Brown, et al.): Bacterial. 177: 6536-6544 (1995)) and KatG from E. coli (Triggs-Raine, B. L. & Loewen, P. C. Gene 52: 121-128 (1987)).
2. Promoters
Plant promoters can be selected to control the expression of the transgene in different plant tissues or organelles for all of which methods are known to those skilled in the art (Gasser & Fraley, Science 244:1293-99 (1989)). In one embodiment, promoters are selected from those of eukaryotic or synthetic origin that are known to yield high levels of expression in plant and algae cytosol. In another embodiment, promoters are selected from those of plant or prokaryotic origin that are known to yield high expression in plastids. In certain embodiments the promoters are inducible. Inducible plant promoters are known in the art.
Suitable constitutive promoters for nuclear-encoded expression include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in U.S. Pat. No. 6,072,050; the core CAMV 355 promoter, (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); and ALS promoter (U.S. Pat. No. 5,659,026). Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142.
“Tissue-preferred” promoters can be used to target a gene expression within a particular tissue such as seed, leaf or root tissue. Tissue-preferred promoters include Yamamoto et al. (1997) Plant J 12(2)255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al (1997) Mol. Gen. Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505.
“Seed-preferred” promoters include both “seed-specific” promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as “seed-germinating” promoters (those promoters active during seed germination). See Thompson et al. (1989) BioEssays 10:108. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphate synthase); and ce1A (cellulose synthase). Gama-zein is a preferred endosperm-specific promoter. Glob-1 is a preferred embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean β-phaseolin, napin β-conglycinin, soybean lectin, cruciferin, oleosin, the Lesquerella hydroxylase promoter, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. Additional seed specific promoters useful for practicing this invention are described in the Examples disclosed herein.
Leaf-specific promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
Root-preferred promoters are known and may be selected from the many available from the literature or isolated de nova from various compatible species. See, for example, Hire et al. (1992) Plant Mol. Biol. 20(2): 207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1):1 1′-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.
Plastid specific promoters include the PrbcL promoter [Allison L. A. et al., EMBO 15: 2802-2809 (1996); Shiina T. et al., Plant Cell 10: 1713-1722 (1998)]; the PpsbA promoter [Agrawal G K, et al., Nucleic Acids Research 29: 1835-1843 (2001)]; the Prrn 16 promoter [Svab Z & Maliga P., Proc. Natl. Acad. Sci. USA 90: 913-917 (1993), Allison L A et al., EMBO 15: 2802-2809 (1996)]; the PaccD promoter (WO97/06250; Hajdukiewicz P T J et al., EMBO J. 16: 4041-4048 (1997)).
Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize 1n2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1 a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. Proc. Natl. Acad. Sci. USA 88:10421-10425 (1991) and McNellis et al. Plant J 14(2):247-257 (1998)) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. Mol. Gen. Genet. 227:229-237 (1991), and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference in their entirety.
In one embodiment, coordinated expression of the three transgenes, phaA, phaB, and phaC, necessary for conversion of acetyl-CoA to PHB is controlled by a seed specific promoter, such as the soybean oleosin promoter (Rowley et al., Biochim Biophys Acta, 1997, 1345, 1-4) or the promoter from the lesquerlla hydroxylase gene (U.S. Pat. No. 6,437,220 B1). In another embodiment, coordinated expression of the three transgenes, phaA, phaB, and phaC, necessary for conversion of acetyl-CoA to PHB is controlled by a promoter active primarily in the biomass plant, such as the maize chlorophyll A/B binding protein promoter (Sullivan et al., Mol. Gen. Genet., 1989, 215, 431-40). It has been previously shown that plants transformed with multi-gene constructs produced higher levels of polymer than plants obtained from crossing single transgene lines (Valentin et al., Int. J. Biol. Macromol., 1999, 25, 303-306; Bohmert et al., Planta, 2000, 211, 841-845).
In one embodiment, the final molecular weight of the polymer produced is controlled by the choice of promoter for expression of the PHA synthase gene. As described in U.S. Pat. No. 5,811,272, high PHA synthase activity will lower polymer molecular weight and low PHA synthase activity will increase polymer molecular weight. In another embodiment, a strong promoter is used for expression of the genes encoding plastid-targeted monomer producing enzymes while a weaker promoter is used to control expression of synthase.
3. Transcription Termination Sequences
At the extreme 3′ end of the transcript of the transgene, a polyadenylation signal can be engineered. A polyadenylation signal refers to any sequence that can result in polyadenylation of the mRNA in the nucleus prior to export of the mRNA to the cytosol, such as the 3′ region of nopaline synthase (Bevan, M., Barnes, W. M., Chilton, M. D. Nucleic Acids Res. 1983, 11, 369-385).
4. Selectable Markers
Genetic constructs may encode a selectable marker to enable selection of plastid transformation events. There are many methods that have been described for the selection of transformed plants [for review see (Miki et al., Journal of Biotechnology, 2004, 107, 193-232) and references incorporated within]. Selectable marker genes that have been used extensively in plants include the neomycin phosphotransferase gene nptII (U.S. Pat. No. 5,034,322, U.S. Pat. No. 5,530,196), hygromycin resistance gene (U.S. Pat. No. 5,668,298), the bar gene encoding resistance to phosphinothricin (U.S. Pat. No. 5,276,268), the expression of aminoglycoside 3″-adenyltransferase (aadA) to confer spectinomycin resistance (U.S. Pat. No. 5,073,675), the use of inhibition resistant 5-enolpyruvyl-3-phosphoshikimate synthetase (U.S. Pat. No. 4,535,060) and methods for producing glyphosate tolerant plants (U.S. Pat. No. 5,463,175; U.S. Pat. No. 7,045,684). Methods of plant selection that do not use antibiotics or herbicides as a selective agent have been previously described and include expression of glucosamine-6-phosphate deaminase to inactive glucosamine in plant selection medium (U.S. Pat. No. 6,444,878) and a positive/negative system that utilizes D-amino acids (Erikson et al., Nat Biotechnol, 2004, 22, 455-8). European Patent Publication No. EP 0 530 129 A1 describes a positive selection system which enables the transformed plants to outgrow the non-transformed lines by expressing a transgene encoding an enzyme that activates an inactive compound added to the growth media. U.S. Pat. No. 5,767,378 describes the use of mannose or xylose for the positive selection of transgenic plants. Methods for positive selection using sorbitol dehydrogenase to convert sorbitol to fructose for plant growth have also been described (WO 2010/102293). Screenable marker genes include the beta-glucuronidase gene (Jefferson et al., 1987, EMBO J. 6: 3901-3907; U.S. Pat. No. 5,268,463) and native or modified green fluorescent protein gene (Cubitt et al., 1995, Trends Biochem. Sci. 20: 448-455; Pan et al., 1996, Plant Physiol. 112: 893-900).
Transformation events can also be selected through visualization of fluorescent proteins such as the fluorescent proteins from the nonbioluminescent Anthozoa species which include DsRed, a red fluorescent protein from the Discosoma genus of coral (Matz et al. (1999), Nat Biotechnol 17: 969-73). An improved version of the DsRed protein has been developed (Bevis and Glick (2002), Nat Biotech 20: 83-87) for reducing aggregation of the protein. Visual selection can also be performed with the yellow fluorescent proteins (YFP) including the variant with accelerated maturation of the signal (Nagai, T. et al. (2002), Nat Biotech 20: 87-90), the blue fluorescent protein, the cyan fluorescent protein, and the green fluorescent protein (Sheen et al. (1995), Plant J 8: 777-84; Davis and Vierstra (1998), Plant Molecular Biology 36: 521-528). A summary of fluorescent proteins can be found in Tzfira et al. (Tzfira et al. (2005), Plant Molecular Biology 57: 503-516) and Verkhusha and Lukyanov (Verkhusha, V. V. and K. A. Lukyanov (2004), Nat Biotech 22: 289-296) whose references are incorporated in entirety. Improved versions of many of the fluorescent proteins have been made for various applications. Use of the improved versions of these proteins or the use of combinations of these proteins for selection of transformants will be obvious to those skilled in the art. It is also practical to simply analyze progeny from transformation events for the presence of the PHB thereby avoiding the use of any selectable marker.
For plastid transformation constructs, a preferred selectable marker is the spectinomycin-resistant allele of the plastid 16S ribosomal RNA gene (Staub J M, Maliga P, Plant Cell 4: 39-45 (1992); Svab Z, Hajdukiewicz P, Maliga P, Proc. Natl., Acad. Sci. USA 87: 8526-8530 (1990)). Selectable markers that have since been successfully used in plastid transformation include the bacterial aadA gene that encodes aminoglycoside adenyltransferase (AadA) conferring spectinomycin and streptomycin resistance (Svab et al., Proc, Natl. Acad. Sci. USA, 1993, 90, 913-917), nptII that encodes aminoglycoside phosphotransferase for selection on kanamycin (Carrer H, Hockenberry T N, Svab Z, Maliga P., Mol. Gen. Genet. 241: 49-56 (1993); Lutz K A, et al., Plant J. 37: 906-913 (2004); Lutz K A, et al., Plant Physiol. 145: 1201-1210 (2007)), aphA6, another aminoglycoside phosphotransferase (Huang F—C, et al, Mol. Genet. Genomics 268: 19-27 (2002)), and chloramphenicol acetyltransferase (Li, W., et al. (2010), Plant Mol Biol, DOI 10.1007/s11103-010-9678-4). Another selection scheme has been reported that uses a chimeric betaine aldehyde dehydrogenase gene (BADH) capable of converting toxic betaine aldehyde to nontoxic glycine betaine (Daniell H, et al., Curr. Genet. 39: 109-116 (2001)).
5. Plastid Targeting Signals
Plastid targeting sequences are known in the art and include the chloroplast small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al, Plant Mal. Biol. 30:769-780 (1996); Schnell et J. Biol. Chem. 266(5):3335-3342 (1991)); 5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al. J. Bioenerg. Biomemb. 22(6):789-810 (1990)); tryptophan synthase (Zhao et al. J. Biol. Chem. 270(11):6081-6087 (1995)); plastocyanin (Lawrence et al. J. Biol. Chem. 272(33):20357-20363 (1997)); chorismate synthase (Schmidt et al. J. Biol. Chem. 268(36):27447-27457 (1993)); and the light harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al. J. Biol. Chem. 263:14996-14999 (1988)). See also Von Heijne et al. Plant Mol. Biol. Rep. 9:104-126 (1991); Clark et al. J. Biol. Chem. 264:17544-17550 (1989); Della-Cioppa et al. Plant Physiol. 84:965-968 (1987); Romer et al. Biochem. Biophys. Res. Commun. 196:1414-1421 (1993); and Shah et al. Science 233:478-481 (1986). Alternative plastid targeting signals have also been described in the following: US 2008/0263728; Miras, S. et al. (2002), J Biol Chem 277(49): 47770-8; Miras, S. et al. (2007), J Biol Chem 282: 29482-29492.
B. Exemplary Host Plants
Plants transformed in accordance with the present disclosure may be monocots or dicots. The transformation of suitable agronomic plant hosts using vectors for nuclear transformation or direct plastid transformation can be accomplished with a variety of methods and plant tissues. Representative plants useful in the methods disclosed herein include the Brassica family including B. napus, B. rapa, B. carinata and B. juncea; industrial oilseeds such as Camelina sativa, Crambe, jatropha, castor; Calendula, Cuphea, Arabidopsis thaliana; maize; soybean; cottonseed; sunflower; palm; coconut; safflower; peanut; mustards including Sinapis alba; sugarcane flax and tobacco, also are useful with the methods disclosed herein. Representative tissues for transformation using these vectors include protoplasts, cells, callus tissue, leaf discs, pollen, and meristems.
C. Methods of Plant Transformation
Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No. 5,563,055; Zhao et al. WO US98/01268), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al. (1995) Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al. Biotechnology 6:923-926 (1988)). Also see Weissinger et al. Ann. Rev. Genet. 22:421-477 (1988); Sanford et al, Particulate Science and Technology 5:27-37 (1987) (onion); Christou et al. Plant Physiol. 87:671-674 (1988) (soybean); McCabe et al. (1988) BioTechnology 6:923-926 (soybean); Finer and McMullen In Vitro Cell Dev. Biol. 27P:175-182 (1991) (soybean); Singh et al. Theor. Appl. Genet. 96:319-324 (1998) (soybean); Dafta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. Proc. Natl. Acad. Sci. USA 85:4305-4309 (1988) (maize); Klein et al. Biotechnology 6:559-563 (1988) (maize); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al. Plant Physiol. 91:440-444 (1988) (maize); Fromm et al. Biotechnology 8:833-839 (1990) (maize); Hooykaas-Van Slogteren et al. Nature 311:763-764 (1984); Bowen et al., U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. Proc. Natl. Acad. Sci. USA 84:5345-5349 (1987) (Liliaceae); De Wet et al. in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp. 197-209 (1985) (pollen); Kaeppler et al. Plant Cell Reports 9:415-418 (1990) and Kaeppler et al. Theor. Appl. Genet. 84:560-566 (1992) (whisker-mediated transformation); D'Halluin et al. Plant Cell 4:1495-1505 (1992) (electroporation); Li et al. Plant Cell Reports 12:250-255 (1993) and Christou and Ford Annals of Botany 75:407-413 (1995) (rice); Osjoda et al. Nature Biotechnology 14:745-750 (1996) (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference in their entirety. References for protoplast transformation and/or gene gun for Agrisoma technology are described in WO 2010/037209. Methods for transforming plant protoplasts are available including transformation using polyethylene glycol (PEG), electroporation, and calcium phosphate precipitation (see for example Potrykus et al., 1985, Mol. Gen. Genet., 199, 183-188; Potrykus et al., 1985, Plant Molecular Biology Reporter, 3, 117-128), Methods for plant regeneration from protoplasts have also been described [Evans et al., in Handbook of Plant Cell Culture, Vol 1, (Macmillan Publishing Co., New York, 1983); Vasil, 1K in Cell Culture and Somatic Cell Genetics (Academic, Orlando, 1984)].
Methods for transformation of plastids such as chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation may be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase (McBride et al., Proc. Natl. Acad. Sci. USA, 1994, 91:7301-7305) or by use of an integrase, such as the phiC31 phage site-specific integrase, to target the gene insertion to a previously inserted phage attachment site (Lutz et al., Plant J, 2004, 37, 906-13). Plastid transformation vectors can be designed such that the transgenes are expressed from a promoter sequence that has been inserted with the transgene during the plastid transformation process or, alternatively, from an endogenous plastidial promoter such that an extension of an existing plastidial operon is achieved (Herz et al., Transgenic Research, 2005, 14, 969-982). An alternative method for plastid transformation as described in WO 2010/061186 wherein RNA produced in the nucleus of a plant cell can be targeted to the plastid genome can also be used to practice the disclosed invention. Inducible gene expression from the plastid genome using a synthetic riboswitch has also been reported (Verhounig et al. (2010), Proc Natl Acad Sci USA 107: 6204-6209). Methods for designing plastid transformation vectors are described by Lutz et al. (Lutz et al., Plant Physiol, 2007, 145, 1201-10).
Recombinase technologies which are useful for producing the disclosed transgenic plants include the cre-lox, FLP/FRT and Gin systems. Methods by which these technologies can be used for the purpose described herein are described for example in (U.S. Pat. No. 5,527,695; Dale And Ow, 1991, Proc. Natl. Acad. Sci. USA 88: 10558-10562; Medberry et al., 1995, Nucleic Acids Res. 23: 485-490).
D. Methods for Reproducing Transgenic Plants
Following transformation by any one of the methods described above, the following procedures can be used to obtain a transformed plant expressing the transgenes: select the plant cells that have been transformed on a selective medium; regenerate the plant cells that have been transformed to produce differentiated plants; select transformed plants expressing the transgene producing the desired level of desired polypeptide(s) in the desired tissue and cellular location.
In plastid transformation procedures, further rounds of regeneration of plants from explants of a transformed plant or tissue can be performed to increase the number of transgenic plastids such that the transformed plant reaches a state of homoplasmy (all plastids contain uniform plastomes containing transgene insert).
The cells that have been transformed may be grown into plants in accordance with conventional techniques. See, for example, McCormick et al, Plant Cell Reports 5:81-84 (1986). These plants may then be grown, and either pollinated with the same transformed variety or different varieties, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that constitutive expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure constitutive expression of the desired phenotypic characteristic has been achieved.
In some scenarios, it may be advantageous to insert a multi-gene pathway into the plant by crossing of lines containing portions of the pathway to produce hybrid plants in which the entire pathway has been reconstructed. This is especially the case when high levels of product in a seed compromises the ability of the seed to germinate or the resulting seedling to survive under normal soil growth conditions. Hybrid lines can be created by crossing a line containing one or more PHB genes with a line containing the other gene(s) needed to complete the PHB biosynthetic pathway. Use of lines that possess cytoplasmic male sterility (Esser, K. et al., 2006, Progress in Botany, Springer Berlin Heidelberg. 67, 31-52) with the appropriate maintainer and restorer lines allows these hybrid lines to be produced efficiently. Cytoplasmic male sterility systems are already available for some Brassicaceae species (Esser, K. et al., 2006, Progress in Botany, Springer Berlin Heidelberg. 67, 31-52). These Brassicaceae species can be used as gene sources to produce cytoplasmic male sterility systems for other oilseeds of interest such as Camelina.

III. Methods for Use

The disclosed genetic constructs can be used to produce industrial oilseed plants for high levels of PHA production. Specifically, PHA is produced in the seed.
The transgenic plants can be grown and harvested. The polyhydroxyalkanoate can be isolated from the oilseeds and the remaining plant material can be used as a feedstock for industrial use, preferably for the production of oleochemicals, energy or for use as feed for animals. The polyhydroxyalkanoate harvested from the plants can then be used to produce plastics, rubber material, coating material, and binders for paints, or as a feedstock for producing chemical derivatives such as hydroxyacids, esters, alkenoic acids or amines. PHA also has several medical applications.
The present invention will be further understood by reference to the following non-limiting examples.

EXAMPLES

Example 1

Design and Construction of Transformation Vectors for Production of PHB in Oilseeds

Five different vectors for seed specific expression of the PHB pathway were constructed containing different seed specific promoters for production of PHB in oilseeds (Table 1). Vector pMBXS490, a pCAMBIA based plasmid (Centre for Application of Molecular Biology to International Agriculture, Canberra, Australia), contains the following gene expression cassettes: (1) an expression cassette for PHA synthase containing the promoter from the soybean oleosin isoform A gene, a DNA fragment encoding the signal peptide of the small subunit of rubisco from pea (P. sativum) and the first 24 amino acids of the mature protein (Cashmore, A. R. 1983, In Genetic Engineering of Plants, pp. 29-38), a DNA fragment encoding a hybrid PHA synthase (PhaC; U.S. Pat. No. 6,316,262) in which the first nine amino acids at the N-terminus of this synthase are derived from the Pseudomonas oleovorans phaC1 gene and the remainder of the synthase coding sequence is derived from Zoogloea ramigera phaC gene, and the 3′ termination sequence from the soybean oleosin isoform A gene; (2) an expression cassette for reductase containing the promoter from the soybean oleosin isoform A gene, a DNA fragment encoding the signal peptide and the first 24 amino acids of the mature protein of the small subunit of rubisco from pea, a DNA fragment encoding a NADPH dependent reductase (PhaB) from Ralstonia eutropha eutropha (Peoples, O. & A. Sinskey, 1989, J. Biol. Chem., 264, 15293-15297), and the 3′ termination sequence from the soybean oleosin isoform A gene; (3) an expression cassette for thiolase containing the promoter from the soybean glycinin (gy1) gene (Iida et al., 1995, Plant Cell Reports, 14, 539-544), a DNA fragment encoding the signal peptide and the first 24 amino acids of the mature protein of the small subunit of rubisco from pea, the phaA gene encoding a β-ketothiolase (PhaA) from Ralstonia eutropha (Peoples, O. & A. Sinskey, 1989, J. Biol. Chem., 264, 15293-15297), and a 3′ termination sequence from the soybean glycinin gene; (4) an expression cassette for DsRed, a protein that can be visualized in seeds by placing them in light of the appropriate wavelength, containing the promoter from the cassava mosaic virus (CMV), a DNA fragment encoding a modified red fluorescent protein from Discosoma sp. (DsRed) in which eleven amino acids have been added to the C-terminus to increase solubility and/or prevent aggregation of the protein, and a termination sequence from the Agrobacterium tumefaciens nopaline synthase gene.

TABLE 1

Summary of transformation vectors
containing seed specific promoters

	Promoter controlling	Selectable or
Plasmid	expression of pha genes	visible marker

pMBXS490	Oleosin	DsRed
pMBXS364	LH	DsRed
pMBXS355	LH	bar
pMBXS491	Napin	DsRed
pMBXS492	Glycinin	DsRed

Promoters are as follows: LH, promoter from the Lesquerella fendleri bifunctional oleate 12-hydroxylase:saturate gene (U.S. Pat. No. 6,437,220 B1); Oleosin, promoter from the soybean oleosin isoform A gene (Rowley and Herman, 1997, Biochim. Biophys. Acta 1345, 1-4); Napin, promoter from the Brassica napes napin gene (Ellenstrom, M. et al., 1996, Plant Molecular Biology, 32: 1019-1027); Glycinin, promoter from the soybean glycinin (gy1) gene (Iida, A. et al., 1995, Plant Cell Reports, 14:539-544).
Vectors pMBXS364, pMBXS355, pMBXS491, and pMBXS492 contain the same PHB pathway genes as pMBXS490 with the exception that the expression of these genes is under the control of different promoters as outlined in Table 1. Vector pMBXS355 contains an expression cassette for the bar gene, encoding phosphinothricin acetyltransferase whose expression is under the control of the 35S promoter. Expression of the bar gene allows selection of transformants based on their resistance to bialaphos. All other vectors in Table 1 contain expression cassettes for DsRed allowing the identification of transgenic seeds under the appropriate wavelength of light.

Example 2

Transformation of Camelina

In preparation for plant transformation experiments, seeds of Camelina sativa cultivar Suneson or Celine were sown directly into 4 inch pots filled with soil (Metro mix) in the greenhouse. Growth conditions were maintained at 24° C. during the day and 18° C. during the night. Plants were grown until flowering. Plants with a number of unopened flower buds were used in ‘floral dip’ transformations.
Agrobacterium strain GV3101 was transformed with the construct of interest using electroporation. A single colony of GV3101 containing the construct of interest was obtained from a freshly streaked plate and was inoculated into 5 mL LB medium. After overnight growth at 28° C., 2 mL of culture was transferred to a 500-mL flask containing 300 mL of LB and incubated overnight at 28° C. Cells were pelleted by centrifugation (6,000 rpm, 20 min), and diluted to an OD600 of ˜0.8 with infiltration medium containing 5% sucrose and 0.05% (v/v) Silwet-L77 (Lehle Seeds, Round Rock, Tex., USA). Camelina plants were transformed by “floral dip” using transformation constructs as follows. Pots containing plants at the flowering stage were placed inside a 460 mm height vacuum desiccator (Bel-Art, Pequannock, N.J., USA). Inflorescences were immersed into the Agrobacterium inoculum contained in a 500-ml beaker. A vacuum (85 kPa) was applied and held for 5 min. Plants were removed from the desiccator and were covered with plastic bags in the dark for 24 h at room temperature. Plants were removed from the bags and returned to normal growth conditions within the greenhouse for seed formation.
To identify Camelina seeds expressing DsRed, fully mature seeds were harvested from transformed plants and placed in a desiccator with anhydrous calcium sulfate as desiccant for at least 2 days prior to screening. DsRed expressing seeds were visualized in a darkroom with a green LumaMax LED flashlight (Lab Safety Supply, Inc., Janesville, Wis.) and a pair of KD's Dark Red glasses (Pacific Coast Sunglasses Inc., Santa Maria, Calif.).
To identify bialaphos resistant seeds, seeds from floral dip transformations were sterilized in 70% ethanol and 10% bleach, and washed in water. Sterilized seeds were placed on germination and selection medium in square Petri dishes. The germination and selection medium contained 10 mg/L bialaphos (Gold BioTechnology, 130178-500) in ½× MS medium, which was made with Murashige & Skoog medium mixture (Caisson Labs, MSP09) at half concentration. The plates were sealed and placed in a growth chamber for germination under a 16-h photoperiod, 3,000 lux light intensity, and temperatures of 23/20° C. at day/night. Seedlings with greenish cotyledons were picked and transferred to soil about six days after initiation of germination.

Example 3

Production of PHB in Seeds of Camelina

In initial transformation experiments with pMBXS490, 24 DsRed positive seeds were isolated. Four of these seeds were sacrificed to determine their PHB content using a previously described gas chromatography/butanolysis technique performed essentially as previously described (Somleva et al., 2008, Plant Biotechnol. J., 663-678). These four seeds contained 19.9, 12.0, 9.8, and 6.4% dwt PHB in the seed. When other seeds from this transformation were planted in soil, seedlings possessed whitish cotyledons and their growth was severely impaired. Only a few T₁seeds with low levels of PHB were capable of germination and survival in soil in a greenhouse. These seedlings were still weak and possessed white or variegated cotyledons.
In transformations of pMBXS355 and pMBXS364, seeds from transformed plants were screened for resistance to bialophos and or visual screening for DsRed, respectively. Despite having the same promoter controlling the expression of the PHB biosynthetic pathway, the maximum PHB production in pMBXS355 (0.54% PHB) was significantly lower than the amount produced by pMBXS364 (3.4%) (Table 2). This is likely due to difficulty in distinguishing between weak pMBXS355 seedlings that produced higher levels of PHB and the non-transformed, bialophos sensitive seedlings.

TABLE 2

Comparison of PHB production in Lines isolated
using bialaphos selection or visual screening

	Selectable or	# of	# of Lines w/	Range of PHB
	Screenable	Lines	PHB in T2	Production
Vector	Marker	Tested	Seeds	(% seed weight)

pMBXS355	Bar¹	204	5	0.05 to 0.54%
pMBXS364	DsRed²	170	85	0.5 to 3.4%

¹Selection of transformants performed by germination of seeds on tissue culture plates containing 10 mg/L bialophos.
²Selection of transformants performed by visual screening for DsRed expression.

In transformations with pMBX491 and pMBX492 containing the PHB genes under the control of the napin and glycinin promoters, respectively, were healthier than transformants obtained from pMBX490 transformations. For pMBX491, T2 seeds were isolated containing 8% PHB in DsRed seeds picked from the segregating population. These seeds possessed a 75% germination rate and a 60% survival rate under greenhouse conditions in soil. The cotyledons after 11 days were chlorotic and the growth of this line was significantly delayed compared to wild-type. For pMBX492, T2 seeds were isolated containing 6.9% PHB in DsRed seeds picked from the segregating population. These seeds possessed a 75% germination rate and a 70% survival rate under greenhouse conditions in soil. After 11 days, the cotyledons and first true leaves of this transformant were green. The growth of this line was somewhat delayed compared to wild-type but faster than the pMBXS491 line.
The 19% dwt PHB produced in a single seed obtained from Camelina plants transformed with construct pMBXS490 was an unexpected result and is the highest level of PHB reported in oilseeds to date. Previous studies with Brassica napus produced up to 7.7% dwt PHB. These seeds were obtained from transformation of Brassica napus using stem segments as the explants and selection of the transformed explants (Fry, J. et al., 1987, 6, 321-325) using glyphosate resistance obtained from expression of a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase. Researchers did not report any germination issues with seeds isolated from the transformed plants [Houmiel et al., 1999, Planta, 209, 547-550; Valentin et al., 1999, Int. J. Biol. Macromol. 25, 303-306].
The use of DsRed as a visual marker in Camelina enabled the identification of high PHB producing seeds that would not have germinated in a typical seed screening procedure where an antibiotic or herbicide selectable marker, such as glyphosate resistance, is employed to provide resistance to the selection agent during seed germination and seedling development in tissue culture medium.

Example 4

Transformation of Brassica napus, Brassica carinata, and Brassica juncea

Transformation of Brassica carinata
Brassica carinata can be transformed using a previously described floral dip method (Shiv et al., 2008, Journal of Plant Biochemistry and Biotechnology 17, 1-4). Briefly constructs of interest are transformed into Agrobacterium strain GV-3101 and cells are grown in liquid medium. Cells are harvested and resuspended in a transformation medium consisting of ½ MS salts, 5% sucrose, and 0.05% Silwet L-77. Brassica carinata plants are grown in a greenhouse until inflorescences develop and approximately 25% of their flowers are opened. Plants are submerged in the prepared Agrobacterium solution for approximately 1 minute, and covered for 24 hours. Plants are returned to the greenhouse and allowed to set seed. Transformed seeds are screened by picking DsRed seeds under the appropriate wavelength of light as described above.
Transformation of Brassica napus
Brassica seeds are surface sterilized in 10% commercial bleach (Javex, Colgate-Palmolive) for 30 min with gentle shaking. The seeds are washed three times in sterile distilled water and placed in germination medium comprising Murashige-Skoog (MS) salts and vitamins, 3% (w/v) sucrose and 0.7% (w/v) phytagar, pH 5.8 at a density of 20 per plate and maintained at 24° C. an a 16 h light/8 h dark photoperiod at a light intensity of 60-80 μEm⁻²s⁻¹for 4-5 days.
Constructs of interest are introduced into Agrobacterium tumefacians strain EHA101 (Hood et. al., 1986, J. Bacteriol. 168: 1291-1301) by electroporation. Prior to transformation of cotyledonary petioles, single colonies of strain EHA101 harboring each construct are grown in 5 ml of minimal medium supplemented with appropriate antibiotics for 48 hr at 28° C. One ml of bacterial suspension was pelleted by centrifugation for 1 min in a microfuge. The pellet was resuspended in 1 ml minimal medium.
For transformation, cotyledons are excised from 4 or in some cases 5 day old seedlings so that they included ˜2 mm of petiole at the base. Individual cotyledons with the cut surface of their petioles are immersed in diluted bacterial suspension for 1 s and immediately embedded to a depth of ˜2 mm in co-cultivation medium, MS medium with 3% (w/v) sucrose and 0.7% phytagar and enriched with 20 μM benzyladenine. The inoculated cotyledons are plated at a density of 10 per plate and incubated under the same growth conditions for 48 h. After co-cultivation, the cotyledons are transferred to regeneration medium comprising MS medium supplemented with 3% sucrose, 20 μM benzyladenine, 0.7% (w/v) phytagar, pH 5.8, 300 mg/L timentinin and 20 mg/L kanamycin sulfate.
After 2-3 weeks regenerant shoots obtained are cut and maintained on “shoot elongation” medium (MS medium containing, 3% sucrose, 300 mg/L timentin, 0.7% (w/v) phytagar, 300 mg/L timentinin and 20 mg/L kanamycin sulfate, pH 5.8) in Magenta jars. The elongated shoots are transferred to “rooting” medium comprising MS medium, 3% sucrose, 2 mg/L indole butyric acid, 0.7% phytagar and 500 mg/L carbenicillin. After roots emerge, plantlets are transferred to potting mix (Redi Earth, W.R. Grace and Co.). The plants are maintained in a misting chamber (75% relative humidity) under the same growth conditions. Plants are allowed to self pollinate to produce seeds. Seeds are screened by visualization of DsRed as described above.
Brassica napus can also be transformed using the floral dip procedure described by Shiv et al. (Shiv et al., 2008, Journal of Plant Biochemistry and Biotechnology 17, 1-4) as described above for Brassica carinata.
Transformation of Brassica juncea
Brassica juncea can be transformed using hypocotyl explants according to the methods described by Barfield and Pua (Barfield and Pua, Plant Cell Reports, 10, 308-314) or Pandian et al. (Pandian, et al., 2006, Plant Molecular Biology Reporter 24: 103a-103i) as follows.
B. juncea seeds are sterilized 2 min in 70% (v/v) ethanol and washed for 20 min in 25% commercial bleach (10 g/L hypochlorite). Seeds are rinsed 3× in sterile water. Surface-sterilized seeds are plated on germination medium (1× MS salts, 1×MS vitamins, 30 g/L sucrose, 500 mg/L MES. pH 5.5) and kept in the cold room for 2 days. Seeds are incubated for 4-6 days at 24° C. under low light (20 μm m⁻¹s⁻¹). Hypocotyl segments are excised and rinsed in 50 mL of callus induction medium (1× MS salts, 1× B5 vitamins, 30 g/L sucrose, 500 mg/L MES, 1.0 mg/L 2.4-D, 1.0 mg/L kinetin pH 5.8) for 30 min without agitation. This procedure is repeated but with agitation on orbital shaker (˜140 g) for 48 h at 24° C. in low light (10 μm m⁻¹s⁻¹).
Agrobacterium can be prepared as follows: Cells of Agrobacterium strain AGL1 (Lazo, G. et al. (1991), Biotechnology, 9: 963-967) containing the construct of interest are grown in 5 mL of LB medium with appropriate antibiotic at 28° C. for 2 days. The 5 mL culture is transferred to 250 mL flask with 45 mL of LB and cultured for 4 h at 28° C. Cells is pelleted and resuspended in BM medium (1× MS salts, 1× B5 vitamins, 30 g/L sucrose, 500 mg/L MES, pH 5.8). The optical density at 600 nm is adjusted to 0.2 with BM medium and used for inoculation.
Explants are cocultivated with Agrobacterium for 20 min after which time the Agrobacterium suspension is removed. Hypocotyl explants are washed once in callus induction medium after which cocultivation proceeds for 48 h with gentle shaking on orbital shaker. After several washes in CIM, explants are transferred to selective shoot-inducing medium (500 mg/L AgNO2, 0.4 mg/L zeatin riboside, 2.0 mg/L benzylamino purine, 0.01 mg/L GA, 200 mg/L Timentin appropriate selection agent and 8 g/L agar added to basal medium) plates for regeneration at 24° C. Root formation is induced on root-inducing medium (0.5×MS salts, 0.5× B5 vitamins, 10 g/L sucrose, 500 mg/L MES, 0.1 mg/L indole-3-butyric acid, 200 mg/L Timentin, appropriate selection agent and S g/L agar, pH 5.8).
Plantlets are transferred to are removed from agar, gently washed, and transferred to potting soil in pots. Plants are grown in a humid environment for a week and then transferred to the greenhouse.

Example 5

Production of Hybrid Lines that are not Capable of Germinating

In previous experiments in Arabidopsis, lower levels of PHB were obtained when lines expressing individual PHB genes were crossed to produce a plant containing the entire PHB biosynthetic pathway (Nawrath, C., Y. Poirier, et al., 1994, Proc. Natl. Acad. Sci. USA 91, 12760-12764) than when multi-gene constructs containing the entire PHB biosynthetic pathway were constructed and transformed (Bohmert, K., I. et al., 2000, Planta 211, 841-845;U.S. Pat. No. 6,448,473). This observation led to the subsequent predominant use of multi-gene constructs for PHB production in plants. However, in some scenarios, it may be advantageous to insert a multi-gene pathway into the plant by crossing of lines containing portions of the pathway to produce hybrid plants in which the entire pathway has been reconstructed. This is especially the case when high levels of product in a seed compromises the ability of the seed to germinate or the resulting seedling to survive under normal soil growth conditions. Hybrid lines can be created by crossing a line containing one or more PHB genes with a line containing the other gene(s) needed to complete the PHB biosynthetic pathway. Use of lines that possess cytoplasmic male sterility (Esser, K. et al., 2006, Progress in Botany, Springer Berlin Heidelberg. 67, 31-52) with the appropriate maintainer and restorer lines allows these hybrid lines to be produced efficiently. Cytoplasmic male sterility systems are already available for some Brassicaceae species (Esser, K. et al., 2006, Progress in Botany, Springer Berlin Heidelberg. 67, 31-52). These Brassicaceae species can be used as gene sources to produce cytoplasmic male sterility systems for other oilseeds of interest such as Camelina. Cytoplasmic male sterility has also been reported upon expression of a β-ketothiolase from the chloroplast genome in tobacco (Ruiz, O. N. and H. Daniell, 2005, Plant Physiol. 138, 1232-1246). Male sterility has also been reported upon expression of the faoA gene encoding the α-subunit of the fatty acid β-oxidation complex from Pseudomonas putida (U.S. Pat. No. 6,586,658).
High PHB producing lines that are not capable of germination can be produced using oilseed lines that possess cytoplasmic male sterility (CMS) controlled by an extranuclear genome (i.e. mitochondria or chloroplast). The male sterile line is typically maintained by crossing with a maintainer line that is genetically identical except that it possesses normal fertile cytoplasm and is therefore male fertile. Transformation of the maintainer line with one or more genes for the PHB biosynthetic pathway and crossing this modified maintainer line with the original male sterile line will produce a male sterile line possessing a portion of the PHB biosynthetic pathway. In this example, insertion of the phaA and phaC genes into the maintainer line and crossing with the original male cytoplasmic sterile line will form a male sterile line containing the phaA and phaC genes.
Fertility can be restored to this line using a “restorer line” that carries the appropriate nuclear restorer genes. Alternatively, the restorer line can be transformed with the remaining genes required to complete the PHB biosynthetic pathway and crossed with the previously created male sterile line containing phaA and phaC to produce a hybrid line containing the entire PHB biosynthetic pathway.
Crosses can be performed in the field by planting multiple rows of the male sterile line, the line that will produce the seed, next to a few rows of the male fertile line. Harvested seed can be used for subsequent plantings or as the PHB containing seed for crushing and extraction. When expression cassettes for the PHB genes in this example are controlled by strong promoters, such as the soybean oleosin promoter, high PHB producing seeds generated in this manner will possess weak seedlings upon germination and will not be able to survive field conditions under normal growth circumstances unless treated with a material that promotes seedling strength/vigor. This adds a level of gene containment.
Cytoplasmic male sterility systems are already available for some Brassicaceae species (Esser, K., 2006, Progress in Botany, Springer Berlin Heidelberg. 67, 31-52). These Brassicaceae species can be used as gene sources to produce cytoplasmic male sterility systems for other oilseeds of interest such as Camelina. Cytoplasmic male sterility has also been reported upon expression of a β-ketothiolase from the chloroplast genome in tobacco (Ruiz, O. N. and H. Daniell, 2005, Plant Physiol. 138, 1232-1246). Overexpression of β-ketothiolase in Camelina to generate a male sterile line and subsequent crossing with a line expressing phaB and phaC could also be used for hybrid seed production.
Male sterile lines have also been produced in Brassica napus by overexpression of the faoA gene from Pseudomonas putida under the control of the phaseolin promoter sequence (U.S. Pat. No. 6,586,658).
Double haploid technology can be used to speed up the breeding process. In the double haploid technique, immature pollen grains (haploids) are exposed to treatments that result in doubling of the existing genetic material resulting in homozygous, true breeding material in a single generation.
The references, patents, and patent applications cited throughout are incorporated by reference where permissible in their entireties.

Vector: pMBXS490
(SEQ ID NO: 1)

1	GGGGATCCGT ACGTAAGTAC GTACTCAAAA TGCCAACAAA TAAAAAAAAA

51	GTTGCTTTAA TAATGCCAAA ACAAATTAAT AAAACACTTA CAACACCGGA

101	TTTTTTTTAA TTAAAATGTG CCATTTAGGA TAAATAGTTA ATATTTTTAA

151	TAATTATTTA AAAAGCCGTA TCTACTAAAA TGATTTTTAT TTGGTTGAAA

201	ATATTAATAT GTTTAAATCA ACACAATCTA TCAAAATTAA ACTAAAAAAA

251	AAATAAGTGT ACGTGGTTAA CATTAGTACA GTAATATAAG AGGAAAATGA

301	GAAATTAAGA AATTGAAAGC GAGTCTAATT TTTAAATTAT GAACCTGCAT

351	ATATAAAAGG AAAGAAAGAA TCCAGGAAGA AAAGAAATGA AACCATGCAT

401	GGTCCCCTCG TCATCACGAG TTTCTGCCAT TTGCAATAGA AACACTGAAA

451	CACCTTTCTC TTTGTCACTT AATTGAGATG CCGAAGCCAC CTCACACCAT

501	GAACTTCATG AGGTGTAGCA CCCAAGGCTT CCATAGCCAT GCATACTGAA

551	GAATGTCTCA AGCTCAGCAC CCTACTTCTG TGACGTGTCC CTCATTCACC

601	TTCCTCTCTT CCCTATAAAT AACCACGCCT CAGGTTCTCC GCTTCACAAC

651	TCAAACATTC TCTCCATTGG TCCTTAAACA CTCATCAGTC ATCACCGCGG

701	CCGCGGAATT CATGGCTTCT ATGATATCCT CTTCCGCTGT GACAACAGTC

751	AGCCGTGCCT CTAGGGGGCA ATCCGCCGCA GTGGCTCCAT TCGGCGGCCT

801	CAAATCCATG ACTGGATTCC CAGTGAAGAA GGTCAACACT GACATTACTT

851	CCATTACAAG CAATGGTGGA AGAGTAAAGT GCATGCAGGT GTGGCCTCCA

901	ATTGGAAAGA AGAAGTTTGA GACTCTTTCC TATTTGCCAC CATTGACGAG

951	AGATTCTAGA GTGACTGACG TTGTCATCGT ATCCGCCGCC CGCACCGCGG

1001	TCGGCAAGTT TGGCGGCTCG CTGGCCAAGA TCCCGGCACC GGAACTGGGT

1051	GCCGTGGTCA TCAAGGCCGC GCTGGAGCGC GCCGGCGTCA AGCCGGAGCA

1101	GGTGAGCGAA GTCATCATGG GCCAGGTGCT GACCGCCGGT TCGGGCCAGA

1151	ACCCCGCACG CCAGGCCGCG ATCAAGGCCG GCCTGCCGGC GATGGTGCCG

1201	GCCATGACCA TCAACAAGGT GTGCGGCTCG GGCCTGAAGG CCGTGATGCT

1251	GGCCGCCAAC GCGATCATGG CGGGCGACGC CGAGATCGTG GTGGCCGGCG

1301	GCCAGGAAAA CATGAGCGCC GCCCCGCACG TGCTGCCGGG CTCGCGCGAT

1351	GGTTTCCGCA TGGGCGATGC CAAGCTGGTC GACACCATGA TCGTCGACGG

1401	CCTGTGGGAC GTGTACAACC AGTACCACAT GGGCATCACC GCCGAGAACG

1451	TGGCCAAGGA ATACGGCATC ACACGCGAGG CGCAGGATGA GTTCGCCGTC

1501	GGCTCGCAGA ACAAGGCCGA AGCCGCGCAG AAGGCCGGCA AGTTTGACGA

1551	AGAGATCGTC CCGGTGCTGA TCCCGCAGCG CAAGGGCGAC CCGGTGGCCT

1601	TCAAGACCGA CGAGTTCGTG CGCCAGGGCG CCACGCTGGA CAGCATGTCC

1651	GGCCTCAAGC CCGCCTTCGA CAAGGCCGGC ACGGTGACCG CGGCCAACGC

1701	CTCGGGCCTG AACGACGGCG CCGCCGCGGT GGTGGTGATG TCGGCGGCCA

1751	AGGCCAAGGA ACTGGGCCTG ACCCCGCTGG CCACGATCAA GAGCTATGCC

1801	AACGCCGGTG TCGATCCCAA GGTGATGGGC ATGGGCCCGG TGCCGGCCTC

1851	CAAGCGCGCC CTGTCGCGCG CCGAGTGGAC CCCGCAAGAC CTGGACCTGA

1901	TGGAGATCAA CGAGGCCTTT GCCGCGCAGG CGCTGGCGGT GCACCAGCAG

1951	ATGGGCTGGG ACACCTCCAA GGTCAATGTG AACGGCGGCG CCATCGCCAT

2001	CGGCCACCCG ATCGGCGCGT CGGGCTGCCG TATCCTGGTG ACGCTGCTGC

2051	ACGAGATGAA GCGCCGTGAC GCGAAGAAGG GCCTGGCCTC GCTGTGCATC

2101	GGCGGCGGCA TGGGCGTGGC GCTGGCAGTC GAGCGCAAAT AACTCGAGGC

2151	GGCCGCAGCC CTTTTTGTAT GTGCTACCCC ACTTTTGTCT TTTTGGCAAT

2201	AGTGCTAGCA ACCAATAAAT AATAATAATA ATAATGAATA AGAAAACAAA

2251	GGCTTTAGCT TGCCTTTTGT TCACTGTAAA ATAATAATGT AAGTACTCTC

2301	TATAATGAGT CACGAAACTT TTGCGGGAAT AAAAGGAGAA ATTCCAATGA

2351	GTTTTCTGTC AAATCTTCTT TTGTCTCTCT CTCTCTCTCT TTTTTTTTTT

2401	TCTTTCTTCT GAGCTTCTTG CAAAACAAAA GGCAAACAAT AACGATTGGT

2451	CCAATGATAG TTAGCTTGAT CGATGATATC TTTAGGAAGT GTTGGCAGGA

2501	CAGGACATGA TGTAGAAGAC TAAAATTGAA AGTATTGCAG ACCCAATAGT

2551	TGAAGATTAA CTTTAAGAAT GAAGACGTCT TATCAGGTTC TTCATGACTT

2601	AAGCTTTAAG AGGAGTCCAC CATGGTAGAT CTGACTAGTA GAAGGTAATT

2651	ATCCAAGATG TAGCATCAAG AATCCAATGT TTACGGGAAA AACTATGGAA

2701	GTATTATGTG AGCTCAGCAA GAAGCAGATC AATATGCGGC ACATATGCAA

2751	CCTATGTTCA AAAATGAAGA ATGTACAGAT ACAAGATCCT ATACTGCCAG

2801	AATACGAAGA AGAATACGTA GAAATTAAGA AAGAAGAACC AGGCGAAGAA

2851	AAGAATCTTG AAGACGTAAG CACTGACGAC AACACTGAAA AGAAGAAGAT

2901	AAGGTCGGTG ATTGTGAAAG AGACATAGAG GACACATGTA AGGTGGAAAA

2951	TGTAAGGGCG GAAAGTAACC TTATCACAAA GGAATCTTAT CCCCCACTAC

3001	TTATCCTTTT ATATTTTTCC GTGTCATTTT TGCCCTTGAG TTTTCCTATA

3051	TAAGGAACCA AGTTCGGCAT TTGTGAAAAC AAGAAAAAAT TGGTGTAAGC

3101	TATTTTCTTT GAAGTACTGA GGATACAACT TCAGAGAAAT TTGTAAGAAA

3151	GTGGATCGAA ACCATGGCCT CCTCCGAGAA CGTCATCACC GAGTTCATGC

3201	GCTTCAAGGT GCGCATGGAG GGCACCGTGA ACGGCCACGA GTTCGAGATC

3251	GAGGGCGAGG GCGAGGGCCG CCCCTACGAG GGCCACAACA CCGTGAAGCT

3301	GAAGGTGACC AAGGGCGGCC CCCTGCCCTT CGCCTGGGAC ATCCTGTCCC

3351	CCCAGTTCCA GTACGGCTCC AAGGTGTACG TGAAGCACCC CGCCGACATC

3401	CCCGACTACA AGAAGCTGTC CTTCCCCGAG GGCTTCAAGT GGGAGCGCGT

3451	GATGAACTTC GAGGACGGCG GCGTGGCGAC CGTGACCCAG GACTCCTCCC

3501	TGCAGGACGG CTGCTTCATC TACAAGGTGA AGTTCATCGG CGTGAACTTC

3551	CCCTCCGACG GCCCCGTGAT GCAGAAGAAG ACCATGGGCT GGGAGGCCTC

3601	CACCGAGCGC CTGTACCCCC GCGACGGCGT GCTGAAGGGC GAGACCCACA

3651	AGGCCCTGAA GCTGAAGGAC GGCGGCCACT ACCTGGTGGA GTTCAAGTCC

3701	ATCTACATGG CCAAGAAGCC CGTGCAGCTG CCCGGCTACT ACTACGTGGA

3751	CGCCAAGCTG GACATCACCT CCCACAACGA GGACTACACC ATCGTGGAGC

3801	AGTACGAGCG CACCGAGGGC CGCCACCACC TGTTCCTGGT ACCAATGAGC

3851	TCTGTCCAAC AGTCTCAGGG TTAATGTCTA TGTATCTTAA ATAATGTTGT

3901	CGGCGATCGT TCAAACATTT GGCAATAAAG TTTCTTAAGA TTGAATCCTG

3951	TTGCCGGTCT TGCGATGATT ATCATATAAT TTCTGTTGAA TTACGTTAAG

4001	CATGTAATAA TTAACATGTA ATGCATGACG TTATTTATGA GATGGGTTTT

4051	TATGATTAGA GTCCCGCAAT TATACATTTA ATACGCGATA GAAAACAAAA

4101	TATAGCGCGC AAACTAGGAT AAATTATCGC GCGCGGTGTC ATCTATGTTA

4151	CTAGATCGGG AATTAAACTA TCAGTGTTTG ACAGGATATA TTGGCGGGTA

4201	AACCTAAGAG AAAAGAGCGT TTATTAGAAT AACGGATATT TAAAAGGGCG

4251	TGAAAAGGTT TATCCGTTCG TCCATTTGTA TGTGCATGCC AACCACAGGG

4301	TTCCCCTCGG GATCAAAGTA CTTTGATCCA ACCCCTCCGC TGCTATAGTG

4351	CAGTCGGCTT CTGACGTTCA GTGCAGCCGT CTTCTGAAAA CGACATGTCG

4401	CACAAGTCCT AAGTTACGCG ACAGGCTGCC GCCCTGCCCT TTTCCTGGCG

4451	TTTTCTTGTC GCGTGTTTTA GTCGCATAAA GTAGAATACT TGCGACTAGA

4501	ACCGGAGACA TTACGCCATG AACAAGAGCG CCGCCGCTGG CCTGCTGGGC

4551	TATGCCCGCG TCAGCACCGA CGACCAGGAC TTGACCAACC AACGGGCCGA

4601	ACTGCACGCG GCCGGCTGCA CCAAGCTGTT TTCCGAGAAG ATCACCGGCA

4651	CCAGGCGCGA CCGCCCGGAG CTGGCCAGGA TGCTTGACCA CCTACGCCCT

4701	GGCGACGTTG TGACAGTGAC CAGGCTAGAC CGCCTGGCCC GCAGCACCCG

4751	CGACCTACTG GACATTGCCG AGCGCATCCA GGAGGCCGGC GCGGGCCTGC

4801	GTAGCCTGGC AGAGCCGTGG GCCGACACCA CCACGCCGGC CGGCCGCATG

4851	GTGTTGACCG TGTTCGCCGG CATTGCCGAG TTCGAGCGTT CCCTAATCAT

4901	CGACCGCACC CGGAGCGGGC GCGAGGCCGC CAAGGCCCGA GGCGTGAAGT

4951	TTGGCCCCCG CCCTACCCTC ACCCCGGCAC AGATCGCGCA CGCCCGCGAG

5001	CTGATCGACC AGGAAGGCCG CACCGTGAAA GAGGCGGCTG CACTGCTTGG

5051	CGTGCATCGC TCGACCCTGT ACCGCGCACT TGAGCGCAGC GAGGAAGTGA

5101	CGCCCACCGA GGCCAGGCGG CGCGGTGCCT TCCGTGAGGA CGCATTGACC

5151	GAGGCCGACG CCCTGGCGGC CGCCGAGAAT GAACGCCAAG AGGAACAAGC

5201	ATGAAACCGC ACCAGGACGG CCAGGACGAA CCGTTTTTCA TTACCGAAGA

5251	GATCGAGGCG GAGATGATCG CGGCCGGGTA CGTGTTCGAG CCGCCCGCGC

5301	ACGTCTCAAC CGTGCGGCTG CATGAAATCC TGGCCGGTTT GTCTGATGCC

5351	AAGCTGGCGG CCTGGCCGGC CAGCTTGGCC GCTGAAGAAA CCGAGCGCCG

5401	CCGTCTAAAA AGGTGATGTG TATTTGAGTA AAACAGCTTG CGTCATGCGG

5451	TCGCTGCGTA TATGATGCGA TGAGTAAATA AACAAATACG CAAGGGGAAC

5501	GCATGAAGGT TATCGCTGTA CTTAACCAGA AAGGCGGGTC AGGCAAGACG

5551	ACCATCGCAA CCCATCTAGC CCGCGCCCTG CAACTCGCCG GGGCCGATGT

5601	TCTGTTAGTC GATTCCGATC CCCAGGGCAG TGCCCGCGAT TGGGCGGCCG

5651	TGCGGGAAGA TCAACCGCTA ACCGTTGTCG GCATCGACCG CCCGACGATT

5701	GACCGCGACG TGAAGGCCAT CGGCCGGCGC GACTTCGTAG TGATCGACGG

5751	AGCGCCCCAG GCGGCGGACT TGGCTGTGTC CGCGATCAAG GCAGCCGACT

5801	TCGTGCTGAT TCCGGTGCAG CCAAGCCCTT ACGACATATG GGCCACCGCC

5851	GACCTGGTGG AGCTGGTTAA GCAGCGCATT GAGGTCACGG ATGGAAGGCT

5901	ACAAGCGGCC TTTGTCGTGT CGCGGGCGAT CAAAGGCACG CGCATCGGCG

5951	GTGAGGTTGC CGAGGCGCTG GCCGGGTACG AGCTGCCCAT TCTTGAGTCC

6001	CGTATCACGC AGCGCGTGAG CTACCCAGGC ACTGCCGCCG CCGGCACAAC

6051	CGTTCTTGAA TCAGAACCCG AGGGCGACGC TGCCCGCGAG GTCCAGGCGC

6101	TGGCCGCTGA AATTAAATCA AAACTCATTT GAGTTAATGA GGTAAAGAGA

6151	AAATGAGCAA AAGCACAAAC ACGCTAAGTG CCGGCCGTCC GAGCGCACGC

6201	AGCAGCAAGG CTGCAACGTT GGCCAGCCTG GCAGACACGC CAGCCATGAA

6251	GCGGGTCAAC TTTCAGTTGC CGGCGGAGGA TCACACCAAG CTGAAGATGT

6301	ACGCGGTACG CCAAGGCAAG ACCATTACCG AGCTGCTATC TGAATACATC

6351	GCGCAGCTAC CAGAGTAAAT GAGCAAATGA ATAAATGAGT AGATGAATTT

6401	TAGCGGCTAA AGGAGGCGGC ATGGAAAATC AAGAACAACC AGGCACCGAC

6451	GCCGTGGAAT GCCCCATGTG TGGAGGAACG GGCGGTTGGC CAGGCGTAAG

6501	CGGCTGGGTT GTCTGCCGGC CCTGCAATGG CACTGGAACC CCCAAGCCCG

6551	AGGAATCGGC GTGACGGTCG CAAACCATCC GGCCCGGTAC AAATCGGCGC

6601	GGCGCTGGGT GATGACCTGG TGGAGAAGTT GAAGGCCGCG CAGGCCGCCC

6651	AGCGGCAACG CATCGAGGCA GAAGCACGCC CCGGTGAATC GTGGCAAGCG

6701	GCCGCTGATC GAATCCGCAA AGAATCCCGG CAACCGCCGG CAGCCGGTGC

6751	GCCGTCGATT AGGAAGCCGC CCAAGGGCGA CGAGCAACCA GATTTTTTCG

6801	TTCCGATGCT CTATGACGTG GGCACCCGCG ATAGTCGCAG CATCATGGAC

6851	GTGGCCGTTT TCCGTCTGTC GAAGCGTGAC CGACGAGCTG GCGAGGTGAT

6901	CCGCTACGAG CTTCCAGACG GGCACGTAGA GGTTTCCGCA GGGCCGGCCG

6951	GCATGGCCAG TGTGTGGGAT TACGACCTGG TACTGATGGC GGTTTCCCAT

7001	CTAACCGAAT CCATGAACCG ATACCGGGAA GGGAAGGGAG ACAAGCCCGG

7051	CCGCGTGTTC CGTCCACACG TTGCGGACGT ACTCAAGTTC TGCCGGCGAG

7101	CCGATGGCGG AAAGCAGAAA GACGACCTGG TAGAAACCTG CATTCGGTTA

7151	AACACCACGC ACGTTGCCAT GCAGCGTACG AAGAAGGCCA AGAACGGCCG

7201	CCTGGTGACG GTATCCGAGG GTGAAGCCTT GATTAGCCGC TACAAGATCG

7251	TAAAGAGCGA AACCGGGCGG CCGGAGTACA TCGAGATCGA GCTAGCTGAT

7301	TGGATGTACC GCGAGATCAC AGAAGGCAAG AACCCGGACG TGCTGACGGT

7351	TCACCCCGAT TACTTTTTGA TCGATCCCGG CATCGGCCGT TTTCTCTACC

7401	GCCTGGCACG CCGCGCCGCA GGCAAGGCAG AAGCCAGATG GTTGTTCAAG

7451	ACGATCTACG AACGCAGTGG CAGCGCCGGA GAGTTCAAGA AGTTCTGTTT

7501	CACCGTGCGC AAGCTGATCG GGTCAAATGA CCTGCCGGAG TACGATTTGA

7551	AGGAGGAGGC GGGGCAGGCT GGCCCGATCC TAGTCATGCG CTACCGCAAC

7601	CTGATCGAGG GCGAAGCATC CGCCGGTTCC TAATGTACGG AGCAGATGCT

7651	AGGGCAAATT GCCCTAGCAG GGGAAAAAGG TCGAAAAGGT CTCTTTCCTG

7701	TGGATGTACC GTACATTGGG AACCCAAAGC CGTACATTGG GAACCGGAAC

7751	CCGTACATTG GGAACCCAAA GCCGTACATT GGGAACCGGT CACACATGTA

7801	AGTGACTGAT ATAAAAGAGA AAGAAGGCCA TTTTTCCGCC TAAAACTCTT

7851	TAAAACTTAT TAAAACTCTT AAAACCCGCC TGGCCTGTGC ATAACTGTCT

7901	GGCCAGCGCA CAGCCGAAGA GCTGCAAAAA GCGCCTACCC TTCGGTCGCT

7951	GCGCTCCCTA CGCCCCGCCG CTTCGCGTCG GCCTATCGCG GCCGCTGGCC

8001	GCTCAAAAAT GGCTGGCCTA CGGCCAGGCA ATCTACCAGG GCGCGGACAA

8051	GCCGCGCCGT CGCCACTCGA CCGCCGGCGC CCACATCAAG GCACCCTGCC

8101	TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG

8151	GAGACGGTCA CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG

8201	TCAGGGCGCG TCAGCGGGTG TTGGCGGGTG TCGGGGCGCA GCCATGACCC

8251	AGTCACGTAG CGATAGCGGA GTGTATACTG GCTTAACTAT GCGGCATCAG

8301	AGCAGATTGT ACTGAGAGTG CACCATATGC GGTGTGAAAT ACCGCACAGA

8351	TGCGTAAGGA GAAAATACCG CATCAGGCGC TCTTCCGCTT CCTCGCTCAC

8401	TGACTCGCTG CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT

8451	CAAAGGCGGT AATACGGTTA TCCACAGAAT CAGGGGATAA CGCAGGAAAG

8501	AACATGTGAG CAAAAGGCCA GCAAAAGGCC AGGAACCGTA AAAAGGCCGC

8551	GTTGCTGGCG TTTTTCCATA GGCTCCGCCC CCCTGACGAG CATCACAAAA

8601	ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT ATAAAGATAC

8651	CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT

8701	GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC

8751	TTTCTCATAG CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC

8801	TCCAAGCTGG GCTGTGTGCA CGAACCCCCC GTTCAGCCCG ACCGCTGCGC

8851	CTTATCCGGT AACTATCGTC TTGAGTCCAA CCCGGTAAGA CACGACTTAT

8901	CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC GAGGTATGTA

8951	GGCTGGCCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG

9001	AAGGACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA

9051	AAAGAGTTGG TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT

9101	GGTTTTTTTG TTTGCAAGCA GCAGATTACG CGCAGAAAAA AAGGATCTCA

9151	AGAAGATCCT TTGATCTTTT CTACGGGGTC TGACGCTCAG TGGAACGAAA

9201	ACTCACGTTA AGGGATTTTG GTCATGCATT CTAGGTACTA AAACAATTCA

9251	TCCAGTAAAA TATAATATTT TATTTTCTCC CAATCAGGCT TGATCCCCAG

9301	TAAGTCAAAA AATAGCTCGA CATACTGTTC TTCCCCGATA TCCTCCCTGA

9351	TCGACCGGAC GCAGAAGGCA ATGTCATACC ACTTGTCCGC CCTGCCGCTT

9401	CTCCCAAGAT CAATAAAGCC ACTTACTTTG CCATCTTTCA CAAAGATGTT

9451	GCTGTCTCCC AGGTCGCCGT GGGAAAAGAC AAGTTCCTCT TCGGGCTTTT

9501	CCGTCTTTAA AAAATCATAC AGCTCGCGCG GATCTTTAAA TGGAGTGTCT

9551	TCTTCCCAGT TTTCGCAATC CACATCGGCC AGATCGTTAT TCAGTAAGTA

9601	ATCCAATTCG GCTAAGCGGC TGTCTAAGCT ATTCGTATAG GGACAATCCG

9651	ATATGTCGAT GGAGTGAAAG AGCCTGATGC ACTCCGCATA CAGCTCGATA

9701	ATCTTTTCAG GGCTTTGTTC ATCTTCATAC TCTTCCGAGC AAAGGACGCC

9751	ATCGGCCTCA CTCATGAGCA GATTGCTCCA GCCATCATGC CGTTCAAAGT

9801	GCAGGACCTT TGGAACAGGC AGCTTTCCTT CCAGCCATAG CATCATGTCC

9851	TTTTCCCGTT CCACATCATA GGTGGTCCCT TTATACCGGC TGTCCGTCAT

9901	TTTTAAATAT AGGTTTTCAT TTTCTCCCAC CAGCTTATAT ACCTTAGCAG

9951	GAGACATTCC TTCCGTATCT TTTACGCAGC GGTATTTTTC GATCAGTTTT

10001	TTCAATTCCG GTGATATTCT CATTTTAGCC ATTTATTATT TCCTTCCTCT

10051	TTTCTACAGT ATTTAAAGAT ACCCCAAGAA GCTAATTATA ACAAGACGAA

10101	CTCCAATTCA CTGTTCCTTG CATTCTAAAA CCTTAAATAC CAGAAAACAG

10151	CTTTTTCAAA GTTGTTTTCA AAGTTGGCGT ATAACATAGT ATCGACGGAG

10201	CCGATTTTGA AACCGCGGTG ATCACAGGCA GCAACGCTCT GTCATCGTTA

10251	CAATCAACAT GCTACCCTCC GCGAGATCAT CCGTGTTTCA AACCCGGCAG

10301	CTTAGTTGCC GTTCTTCCGA ATAGCATCGG TAACATGAGC AAAGTCTGCC

10351	GCCTTACAAC GGCTCTCCCG CTGACGCCGT CCCGGACTGA TGGGCTGCCT

10401	GTATCGAGTG GTGATTTTGT GCCGAGCTGC CGGTCGGGGA GCTGTTGGCT

10451	GGCTGGTGGC AGGATATATT GTGGTGTAAA CAAATTGACG CTTAGACAAC

10501	TTAATAACAC ATTGCGGACG TTTTTAATGT ACTGAATTAA CGCCGAATTA

10551	ATTCCTAGGC CACCATGTTG GGCCCGGGGC GCGCCGTACG TAGTGTTTAT

10601	CTTTGTTGCT TTTCTGAACA ATTTATTTAC TATGTAAATA TATTATCAAT

10651	GTTTAATCTA TTTTAATTTG CACATGAATT TTCATTTTAT TTTTACTTTA

10701	CAAAACAAAT AAATATATAT GCAAAAAAAT TTACAAACGA TGCACGGGTT

10751	ACAAACTAAT TTCATTAAAT GCTAATGCAG ATTTTGTGAA GTAAAACTCC

10801	AATTATGATG AAAAATACCA CCAACACCAC CTGCGAAACT GTATCCCAAC

10851	TGTCCTTAAT AAAAATGTTA AAAAGTATAT TATTCTCATT TGTCTGTCAT

10901	AATTTATGTA CCCCACTTTA ATTTTTCTGA TGTACTAAAC CGAGGGCAAA

10951	CTGAAACCTG TTCCTCATGC AAAGCCCCTA CTCACCATGT ATCATGTACG

11001	TGTCATCACC CAACAACTCC ACTTTTGCTA TATAACAACA CCCCCGTCAC

11051	ACTCTCCCTC TCTAACACAC ACCCCACTAA CAATTCCTTC ACTTGCAGCA

11101	CTGTTGCATC ATCATCTTCA TTGCAAAACC CTAAACTTCA CCTTCAACCG

11151	CGGCCGCATG GCTTCTATGA TATCCTCTTC CGCTGTGACA ACAGTCAGCC

11201	GTGCCTCTAG GGGGCAATCC GCCGCAGTGG CTCCATTCGG CGGCCTCAAA

11251	TCCATGACTG GATTCCCAGT GAAGAAGGTC AACACTGACA TTACTTCCAT

11301	TACAAGCAAT GGTGGAAGAG TAAAGTGCAT GCAGGTGTGG CCTCCAATTG

11351	GAAAGAAGAA GTTTGAGACT CTTTCCTATT TGCCACCATT GACGAGAGAT

11401	TCTAGAGTGA GTAACAAGAA CAACGATGAG CTGCAGTGGC AATCCTGGTT

11451	CAGCAAGGCG CCCACCACCG AGGCGAACCC GATGGCCACC ATGTTGCAGG

11501	ATATCGGCGT TGCGCTCAAA CCGGAAGCGA TGGAGCAGCT GAAAAACGAT

11551	TATCTGCGTG ACTTCACCGC GTTGTGGCAG GATTTTTTGG CTGGCAAGGC

11601	GCCAGCCGTC AGCGACCGCC GCTTCAGCTC GGCAGCCTGG CAGGGCAATC

11651	CGATGTCGGC CATCATGTCC GCATCTTACC TGCTCAACGC CAAATTCCTC

11701	AGTGCCATGG TGGAGGCGGT GGACACCGCA CCCCAGCAAA AGCAGAAAAT

11751	ACGCTTTGCC GTGCAGCAGG TGATTGATGC CATGTCGCCC GCGAACTTCC

11801	TCGCCACCAA CCCGGAAGCG CAGCAAAAAC TGATTGAAAC CAAGGGCGAG

11851	AGCCTGACGC GTGGCCTGGT CAATATGCTG GGCGATATCA ACAAGGGCCA

11901	TATCTCGCTG TCGGACGAAT CGGCCTTTGA AGTGGGCCGC AACCTGGCCA

11951	TTACCCCGGG CACCGTGATT TACGAAAATC CGCTGTTCCA GCTGATCCAG

12001	TACACGCCGA CCACGCCGAC GGTCAGCCAG CGCCCGCTGT TGATGGTGCC

12051	GCCGTGCATC AACAAGTTCT ACATCCTCGA CCTTCAACCG GAAAATTCGC

12101	TGGTGCGCTA CGCGGTGGAG CAGGGCAACA CCGTGTTCCT GATCTCGTGG

12151	AGCAATCCGG ACAAGTCGCT GGCCGGCACC ACCTGGGACG ACTACGTGGA

12201	GCAGGGCGTG ATCGAAGCGA TCCGCATCGT CCAGGACGTC AGCGGCCAGG

12251	ACAAGCTGAA CATGTTCGGC TTCTGCGTGG GCGGCACCAT CGTTGCCACC

12301	GCACTGGCGG TACTGGCGGC GCGTGGCCAG CACCCGGCGG CCAGCCTGAC

12351	CCTGCTGACC ACCTTCCTCG ACTTCAGCGA CACCGGCGTG CTCGACGTCT

12401	TCGTCGATGA AACCCAGGTC GCGCTGCGTG AACAGCAATT GCGCGATGGC

12451	GGCCTGATGC CGGGCCGTGA CCTGGCCTCG ACCTTCTCGA GCCTGCGTCC

12501	GAACGACCTG GTATGGAACT ATGTGCAGTC GAACTACCTC AAAGGCAATG

12551	AGCCGGCGGC GTTTGACCTG CTGTTCTGGA ATTCGGACAG CACCAATTTG

12601	CCGGGCCCGA TGTTCTGCTG GTACCTGCGC AACACCTACC TGGAAAACAG

12651	CCTGAAAGTG CCGGGCAAGC TGACGGTGGC CGGCGAAAAG ATCGACCTCG

12701	GCCTGATCGA CGCCCCGGCC TTCATCTACG GTTCGCGCGA AGACCACATC

12751	GTGCCGTGGA TGTCGGCGTA CGGTTCGCTC GACATCCTCA ACCAGGGCAA

12801	GCCGGGCGCC AACCGCTTCG TGCTGGGCGC GTCCGGCCAT ATCGCCGGCG

12851	TGATCAACTC GGTGGCCAAG AACAAGCGCA GCTACTGGAT CAACGACGGT

12901	GGCGCCGCCG ATGCCCAGGC CTGGTTCGAT GGCGCGCAGG AAGTGCCGGG

12951	CAGCTGGTGG CCGCAATGGG CCGGGTTCCT GACCCAGCAT GGCGGCAAGA

13001	AGGTCAAGCC CAAGGCCAAG CCCGGCAACG CCCGCTACAC CGCGATCGAG

13051	GCGGCGCCCG GCCGTTACGT CAAAGCCAAG GGCTGAGCGG CCGCTGAGTA

13101	ATTCTGATAT TAGAGGGAGC ATTAATGTGT TGTTGTGATG TGGTTTATAT

13151	GGGGAAATTA AATAAATGAT GTATGTACCT CTTGCCTATG TAGGTTTGTG

13201	TGTTTTGTTT TGTTGTCTAG CTTTGGTTAT TAAGTAGTAG GGACGTTCGT

13251	TCGTGTCTCA AAAAAAGGGG TACTACCACT CTGTAGTGTA TATGGATGCT

13301	GGAAATCAAT GTGTTTTGTA TTTGTTCACC TCCATTGTTG AATTCAATGT

13351	CAAATGTGTT TTGCGTTGGT TATGTGTAAA ATTACTATCT TTCTCGTCCG

13401	ATGATCAAAG TTTTAAGCAA CAAAACCAAG GGTGAAATTT AAACTGTGCT

13451	TTGTTGAAGA TTCTTTTATC ATATTGAAAA TCAAATTACT AGCAGCAGAT

13501	TTTACCTAGC ATGAAATTTT ATCAACAGTA CAGCACTCAC TAACCAAGTT

13551	CCAAACTAAG ATGCGCCATT AACATCAGCC AATAGGCATT TTCAGCAAGG

13601	CGCGCCCGCG CCGATGTATG TGACAACCCT CGGGATTGTT GATTTATTTC

13651	AAAACTAAGA GTTTTTGTCT TATTGTTCTC GTCTATTTTG GATATCAATC

13701	TTAGTTTTAT ATCTTTTCTA GTTCTCTACG TGTTAAATGT TCAACACACT

13751	AGCAATTTGG CCTGCCAGCG TATGGATTAT GGAACTATCA AGTCTGTGAC

13801	GCGCCGTACG TAGTGTTTAT CTTTGTTGCT TTTCTGAACA ATTTATTTAC

13851	TATGTAAATA TATTATCAAT GTTTAATCTA TTTTAATTTG CACATGAATT

13901	TTCATTTTAT TTTTACTTTA CAAAACAAAT AAATATATAT GCAAAAAAAT

13951	TTACAAACGA TGCACGGGTT ACAAACTAAT TTCATTAAAT GCTAATGCAG

14001	ATTTTGTGAA GTAAAACTCC AATTATGATG AAAAATACCA CCAACACCAC

14051	CTGCGAAACT GTATCCCAAC TGTCCTTAAT AAAAATGTTA AAAAGTATAT

14101	TATTCTCATT TGTCTGTCAT AATTTATGTA CCCCACTTTA ATTTTTCTGA

14151	TGTACTAAAC CGAGGGCAAA CTGAAACCTG TTCCTCATGC AAAGCCCCTA

14201	CTCACCATGT ATCATGTACG TGTCATCACC CAACAACTCC ACTTTTGCTA

14251	TATAACAACA CCCCCGTCAC ACTCTCCCTC TCTAACACAC ACCCCACTAA

14301	CAATTCCTTC ACTTGCAGCA CTGTTGCATC ATCATCTTCA TTGCAAAACC

14351	CTAAACTTCA CCTTCAACCG CGGCCGCATG GCTTCTATGA TATCCTCTTC

14401	CGCTGTGACA ACAGTCAGCC GTGCCTCTAG GGGGCAATCC GCCGCAGTGG

14451	CTCCATTCGG CGGCCTCAAA TCCATGACTG GATTCCCAGT GAAGAAGGTC

14501	AACACTGACA TTACTTCCAT TACAAGCAAT GGTGGAAGAG TAAAGTGCAT

14551	GCAGGTGTGG CCTCCAATTG GAAAGAAGAA GTTTGAGACT CTTTCCTATT

14601	TGCCACCATT GACGAGAGAT TCTAGAGTGA CTCAGCGCAT TGCGTATGTG

14651	ACCGGCGGCA TGGGTGGTAT CGGAACCGCC ATTTGCCAGC GGCTGGCCAA

14701	GGATGGCTTT CGTGTGGTGG CCGGTTGCGG CCCCAACTCG CCGCGCCGCG

14751	AAAAGTGGCT GGAGCAGCAG AAGGCCCTGG GCTTCGATTT CATTGCCTCG

14801	GAAGGCAATG TGGCTGACTG GGACTCGACC AAGACCGCAT TCGACAAGGT

14851	CAAGTCCGAG GTCGGCGAGG TTGATGTGCT GATCAACAAC GCCGGTATCA

14901	CCCGCGACGT GGTGTTCCGC AAGATGACCC GCGCCGACTG GGATGCGGTG

14951	ATCGACACCA ACCTGACCTC GCTGTTCAAC GTCACCAAGC AGGTGATCGA

15001	CGGCATGGCC GACCGTGGCT GGGGCCGCAT CGTCAACATC TCGTCGGTGA

15051	ACGGGCAGAA GGGCCAGTTC GGCCAGACCA ACTACTCCAC CGCCAAGGCC

15101	GGCCTGCATG GCTTCACCAT GGCACTGGCG CAGGAAGTGG CGACCAAGGG

15151	CGTGACCGTC AACACGGTCT CTCCGGGCTA TATCGCCACC GACATGGTCA

15201	AGGCGATCCG CCAGGACGTG CTCGACAAGA TCGTCGCGAC GATCCCGGTC

15251	AAGCGCCTGG GCCTGCCGGA AGAGATCGCC TCGATCTGCG CCTGGTTGTC

15301	GTCGGAGGAG TCCGGTTTCT CGACCGGCGC CGACTTCTCG CTCAACGGCG

15351	GCCTGCATAT GGGCTGAGCG GCCGCTGAGT AATTCTGATA TTAGAGGGAG

15401	CATTAATGTG TTGTTGTGAT GTGGTTTATA TGGGGAAATT AAATAAATGA

15451	TGTATGTACC TCTTGCCTAT GTAGGTTTGT GTGTTTTGTT TTGTTGTCTA

15501	GCTTTGGTTA TTAAGTAGTA GGGACGTTCG TTCGTGTCTC AAAAAAAGGG

15551	GTACTACCAC TCTGTAGTGT ATATGGATGC TGGAAATCAA TGTGTTTTGT

15601	ATTTGTTCAC CTCCATTGTT GAATTCAATG TCAAATGTGT TTTGCGTTGG

15651	TTATGTGTAA AATTACTATC TTTCTCGTCC GATGATCAAA GTTTTAAGCA

15701	ACAAAACCAA GGGTGAAATT TAAACTGTGC TTTGTTGAAG ATTCTTTTAT

15751	CATATTGAAA ATCAAATTAC TAGCAGCAGA TTTTACCTAG CATGAAATTT

15801	TATCAACAGT ACAGCACTCA CTAACCAAGT TCCAAACTAA GATGCGCCAT

15851	TAACATCAGC CAATAGGCAT TTTCAGCAAG GCGCGTAA

pMBXS364
(SEQ ID NO: 2)

1	CATGCCAACC ACAGGGTTCC CCTCGGGATC AAAGTACTTT GATCCAACCC

51	CTCCGCTGCT ATAGTGCAGT CGGCTTCTGA CGTTCAGTGC AGCCGTCTTC

101	TGAAAACGAC ATGTCGCACA AGTCCTAAGT TACGCGACAG GCTGCCGCCC

151	TGCCCTTTTC CTGGCGTTTT CTTGTCGCGT GTTTTAGTCG CATAAAGTAG

201	AATACTTGCG ACTAGAACCG GAGACATTAC GCCATGAACA AGAGCGCCGC

251	CGCTGGCCTG CTGGGCTATG CCCGCGTCAG CACCGACGAC CAGGACTTGA

301	CCAACCAACG GGCCGAACTG CACGCGGCCG GCTGCACCAA GCTGTTTTCC

351	GAGAAGATCA CCGGCACCAG GCGCGACCGC CCGGAGCTGG CCAGGATGCT

401	TGACCACCTA CGCCCTGGCG ACGTTGTGAC AGTGACCAGG CTAGACCGCC

451	TGGCCCGCAG CACCCGCGAC CTACTGGACA TTGCCGAGCG CATCCAGGAG

501	GCCGGCGCGG GCCTGCGTAG CCTGGCAGAG CCGTGGGCCG ACACCACCAC

551	GCCGGCCGGC CGCATGGTGT TGACCGTGTT CGCCGGCATT GCCGAGTTCG

601	AGCGTTCCCT AATCATCGAC CGCACCCGGA GCGGGCGCGA GGCCGCCAAG

651	GCCCGAGGCG TGAAGTTTGG CCCCCGCCCT ACCCTCACCC CGGCACAGAT

701	CGCGCACGCC CGCGAGCTGA TCGACCAGGA AGGCCGCACC GTGAAAGAGG

751	CGGCTGCACT GCTTGGCGTG CATCGCTCGA CCCTGTACCG CGCACTTGAG

801	CGCAGCGAGG AAGTGACGCC CACCGAGGCC AGGCGGCGCG GTGCCTTCCG

851	TGAGGACGCA TTGACCGAGG CCGACGCCCT GGCGGCCGCC GAGAATGAAC

901	GCCAAGAGGA ACAAGCATGA AACCGCACCA GGACGGCCAG GACGAACCGT

951	TTTTCATTAC CGAAGAGATC GAGGCGGAGA TGATCGCGGC CGGGTACGTG

1001	TTCGAGCCGC CCGCGCACGT CTCAACCGTG CGGCTGCATG AAATCCTGGC

1051	CGGTTTGTCT GATGCCAAGC TGGCGGCCTG GCCGGCCAGC TTGGCCGCTG

1101	AAGAAACCGA GCGCCGCCGT CTAAAAAGGT GATGTGTATT TGAGTAAAAC

1151	AGCTTGCGTC ATGCGGTCGC TGCGTATATG ATGCGATGAG TAAATAAACA

1201	AATACGCAAG GGGAACGCAT GAAGGTTATC GCTGTACTTA ACCAGAAAGG

1251	CGGGTCAGGC AAGACGACCA TCGCAACCCA TCTAGCCCGC GCCCTGCAAC

1301	TCGCCGGGGC CGATGTTCTG TTAGTCGATT CCGATCCCCA GGGCAGTGCC

1351	CGCGATTGGG CGGCCGTGCG GGAAGATCAA CCGCTAACCG TTGTCGGCAT

1401	CGACCGCCCG ACGATTGACC GCGACGTGAA GGCCATCGGC CGGCGCGACT

1451	TCGTAGTGAT CGACGGAGCG CCCCAGGCGG CGGACTTGGC TGTGTCCGCG

1501	ATCAAGGCAG CCGACTTCGT GCTGATTCCG GTGCAGCCAA GCCCTTACGA

1551	CATATGGGCC ACCGCCGACC TGGTGGAGCT GGTTAAGCAG CGCATTGAGG

1601	TCACGGATGG AAGGCTACAA GCGGCCTTTG TCGTGTCGCG GGCGATCAAA

1651	GGCACGCGCA TCGGCGGTGA GGTTGCCGAG GCGCTGGCCG GGTACGAGCT

1701	GCCCATTCTT GAGTCCCGTA TCACGCAGCG CGTGAGCTAC CCAGGCACTG

1751	CCGCCGCCGG CACAACCGTT CTTGAATCAG AACCCGAGGG CGACGCTGCC

1801	CGCGAGGTCC AGGCGCTGGC CGCTGAAATT AAATCAAAAC TCATTTGAGT

1851	TAATGAGGTA AAGAGAAAAT GAGCAAAAGC ACAAACACGC TAAGTGCCGG

1901	CCGTCCGAGC GCACGCAGCA GCAAGGCTGC AACGTTGGCC AGCCTGGCAG

1951	ACACGCCAGC CATGAAGCGG GTCAACTTTC AGTTGCCGGC GGAGGATCAC

2001	ACCAAGCTGA AGATGTACGC GGTACGCCAA GGCAAGACCA TTACCGAGCT

2051	GCTATCTGAA TACATCGCGC AGCTACCAGA GTAAATGAGC AAATGAATAA

2101	ATGAGTAGAT GAATTTTAGC GGCTAAAGGA GGCGGCATGG AAAATCAAGA

2151	ACAACCAGGC ACCGACGCCG TGGAATGCCC CATGTGTGGA GGAACGGGCG

2201	GTTGGCCAGG CGTAAGCGGC TGGGTTGTCT GCCGGCCCTG CAATGGCACT

2251	GGAACCCCCA AGCCCGAGGA ATCGGCGTGA CGGTCGCAAA CCATCCGGCC

2301	CGGTACAAAT CGGCGCGGCG CTGGGTGATG ACCTGGTGGA GAAGTTGAAG

2351	GCCGCGCAGG CCGCCCAGCG GCAACGCATC GAGGCAGAAG CACGCCCCGG

2401	TGAATCGTGG CACGCGGCCG CTGATCGAAT CCGCAAAGAA TCCCGGCAAC

2451	CGCCGGCAGC CGGTGCGCCG TCGATTAGGA AGCCGCCCAA GGGCGACGAG

2501	CAACCAGATT TTTTCGTTCC GATGCTCTAT GACGTGGGCA CCCGCGTCAG

2551	TCGCAGCATC ATGGACGTGG CCGTTTTCCG TCTGTCGAAG CGTGACCGAC

2601	GAGCTGGCGA GGTGATCCGC TACGAGCTTC CAGACGGGCA CGTAGAGGTT

2651	TCCGCAGGGC CGGCCGGCAT GGCCAGTGTG TGGGATTACG ACCTGGTACT

2701	GATGGCGGTT TCCCATCTAA CCGAATCCAT GAACCGATAC CGGGAAGGGA

2751	AGGGAGACAA GCCCGGCCGC GTGTTCCGTC CACACGTTGC GGACGTACTC

2801	AAGTTCTGCC GGCGAGCCGA TGGCGGAAAG CAGAAAGACG ACCTGGTAGA

2851	AACCTGCATT CGGTTAAACA CCACGCACGT TGCCATGCAG CGTACGAAGA

2901	AGGCCAAGAA CGGCCGCCTG GTGACGGTAT CCGAGGGTGA AGCCTTGATT

2951	AGCCGCTACA AGATCGTAAA GAGCGAAACC GGGCGGCCGG AGTACATCGA

3001	GATCGAGCTA GCTGATTGGA TGTACCGCGA GATCACAGAA GGCAAGAACC

3051	CGGACGTGCT GACGGTTCAC CCCGATTACT TTTTGATCGA TCCCGGCATC

3101	GGCCGTTTTC TCTACCGCCT GGCACGCCGC GCCGCAGGCA AGGCAGAAGC

3151	CAGATGGTTG TTCAAGACGA TCTACGAACG CAGTGGCAGC GCCGGAGAGT

3201	TCAAGAAGTT CTGTTTCACC GTGCGCAAGC TGATCGGGTC AAATGACCTG

3251	CCGGAGTACG ATTTGAAGGA GGAGGCGGGG CAGGCTGGCC CGATCCTAGT

3301	CATGCGCTAC CGCAACCTGA TCGAGGGCGA AGCATCCGCC GGTTCCTAAT

3351	GTACGGAGCA GATGCTAGGG CAAATTGCCC TAGCAGGGGA AAAAGGTCGA

3401	AAAGGTCTCT TTCCTGTGGA TAGCACGTAC ATTGGGAACC CAAAGCCGTA

3451	CATTGGGAAC CGGAACCCGT ACATTGGGAA CCCAAAGCCG TACATTGGGA

3501	ACCGGTCACA CATGTAAGTG ACTGATATAA AAGAGAAAAA AGGCGATTTT

3551	TCCGCCTAAA ACTCTTTAAA ACTTATTAAA ACTCTTAAAA CCCGCCTGGC

3601	CTGTGCATAA CTGTCTGGCC AGCCCACAGC CGAAGAGCTG CAAAAAGCGC

3651	CTACCCTTCG GTCGCTGCGC TCCCTACGCC CCGCCGCTTC GCGTCGGCCT

3701	ATCGCGGCCG CTGGCCGCTC AAAAATGGCT GGCCTACGGC CAGGCAATCT

3751	ACCAGGGCGC GGACAAGCCG CGCCGTCGCC ACTCGACCGC CGGCGCCCAC

3801	ATCAAGGCAC CCTGCCTCGC GCGTTTCGGT GATGACGGTG AAAACCTCTG

3851	ACACATGCAG CTCCCGGAGA CGGTCACAGC TTGTCTGTAA GCGGATGCCG

3901	GGAGCAGACA AGCCCGTCAG GGCGCGTCAG CGGGTGTTGG CGGGTGTCGG

3951	GGCGCAGCCA TGACCCAGTC ACGTAGCGAT AGCGGAGTGT ATACTGGCTT

4001	AACTATGCGG CATCAGAGCA GATTGTACTG AGAGTGCACC ATATGCGGTG

4051	TGAAATACCG CACAGATGCG TAAGGAGAAA ATACCGCATC AGGCGCTCTT

4101	CCGCTTCCTC GCTCACTGAC TCGCTGCGCT CGGTCGTTCG GCTGCGGCGA

4151	GCGGTATCAG CTCACTCAAA GGCGGTAATA CGGTTATCCA CAGAATCAGG

4201	GGATAACGCA GGAAAGAACA TGTGAGCAAA AGGCCAGCAA AAGGCCAGGA

4251	ACCGTAAAAA GGCCGCGTTG CTGGCGTTTT TCCATAGGCT CCGCCCCCCT

4301	GACGAGCATC ACAAAAATCG ACGCTCAAGT CAGAGGTGGC GAAACCCGAC

4351	AGGACTATAA AGATACCAGG CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT

4401	CTCCTGTTCC GACCCTGCCG CTTACCGGAT ACCTGTCCGC CTTTCTCCCT

4451	TCGGGAAGCG TGGCGCTTTC TCATAGCTCA CGCTGTAGGT ATCTCAGTTC

4501	GGTGTAGGTC GTTCGCTCCA AGCTGGGCTG TGTGCACGAA CCCCCCGTTC

4551	AGCCCGACCG CTGCGCCTTA TCCGGTAACT ATCGTCTTGA GTCCAACCCG

4601	GTAAGACACG ACTTATCGCC ACTGGCAGCA GCCACTGGTA ACAGGATTAG

4651	CAGAGCGAGG TATGTAGGCG GTGCTACAGA GTTCTTGAAG TGGTGGCCTA

4701	ACTACGGCTA CACTAGAAGG ACAGTATTTG GTATCTGCGC TCTGCTGAAG

4751	CCAGTTACCT TCGGAAAAAG AGTTGGTAGC TCTTGATCCG GCAAACAAAC

4801	CACCGCTGGT AGCGGTGGTT TTTTTGTTTG CAAGCAGCAG ATTACGCGCA

4851	GAAAAAAAGG ATCTCAAGAA GATCCTTTGA TCTTTTCTAC GGGGTCTGAC

4901	GCTCAGTGGA ACGAAAACTC ACGTTAAGGG ATTTTGGTCA TGCATTCTAG

4951	GTACTAAAAC AATTCATCCA GTAAAATATA ATATTTTATT TTCTCCCAAT

5001	CAGGCTTGAT CCCCAGTAAG TCAAAAAATA GCTCGACATA CTGTTCTTCC

5051	CCGATATCCT CCCTGATCGA CCGGACGCAG AAGGCAATGT CATACCACTT

5101	GTCCGCCCTG CCGCTTCTCC CAAGATCAAT AAAGCCACTT ACTTTGCCAT

5151	CTTTCACAAA GATGTTGCTG TCTCCCAGGT CGCCGTGGGA AAAGACAAGT

5201	TCCTCTTCGG GCTTTTCCGT CTTTAAAAAA TCATACAGCT CGCGCGGATC

5251	TTTAAATGGA GTGTCTTCTT CCCAGTTTTC GCAATCCACA TCGGCCAGAT

5301	CGTTATTCAG TAAGTAATCC AATTCGGCTA AGCGGCTGTC TAAGCTATTC

5351	GTATAGGGAC AATCCGATAT GTCGATGGAG TGAAAGAGCC TGATGCACTC

5401	CGCATACAGC TCGATAATCT TTTCAGGGCT TTGTTCATCT TCATACTCTT

5451	CCGAGCAAAG GACGCCATCG GCCTCACTCA TGAGCAGATT GCTCCAGCCA

5501	TCATGCCGTT CAAAGTGCAG GACCTTTGGA ACAGGCAGCT TTCCTTCCAG

5551	CCATAGCATC ATGTCCTTTT CCCGTTCCAC ATCATAGGTG GTCCCTTTAT

5601	ACCGGCTGTC CGTCATTTTT AAATATAGGT TTTCATTTTC TCCCACCAGC

5651	TTATATACCT TAGCAGGAGA CATTCCTTCC GTATCTTTTA CGCAGCGGTA

5701	TTTTTCGATC AGTTTTTTCA ATTCCGGTGA TATTCTCATT TTAGCCATTT

5751	ATTATTTCCT TCCTCTTTTC TACAGTATTT AAAGATACCC CAAGAAGCTA

5801	ATTATAACAA GACGAACTCC AATTCACTGT TCCTTGCATT CTAAAACCTT

5851	AAATACCAGA AAACAGCTTT TTCAAAGTTG TTTTCAAAGT TGGCGTATAA

5901	CATAGTATCG ACGGAGCCGA TTTTGAAACC GCGGTGATCA CAGGCAGCAA

5951	CGCTCTGTCA TCGTTACAAT CAACATGCTA CCCTCCGCGA GATCATCCGT

6001	GTTTCAAACC CGGCAGCTTA GTTGCCGTTC TTCCGAATAG CATCGGTAAC

6051	ATGAGCAAAG TCTGCCGCCT TACAACGGCT CTCCCGCTGA CGCCGTCCCG

6101	GACTGATGGG CTGCCTGTAT CGAGTGGTGA TTTTGTGCCG AGCTGCCGGT

6151	CGGGGAGCTG TTGGCTGGCT GGTGGCAGGA TATATTGTGG TGTAAACAAA

6201	TTGACGCTTA GACAACTTAA TAACACATTG CGGACGTTTT TAATGTACTG

6251	AATTAACGCC GAATTAATTC GGGGGATCTG GATTTTAGTA CTGGATTTTG

6301	GTTTTAGGAA TTAGAAATTT TATTGATAGA AGTATTTTAC AAATACAAAT

6351	ACATACTAAG GGTTTCTTAT ATGCTCAACA CATGAGCGAA ACCCTATAGG

6401	AACCCTAATT CCCTTATCTG GGAACTACTC ACACATTTTT ATGGAGAAAC

6451	TCGAGTTAAC CCTGAGACTG TTGGACAGAG CTCATTGGTA CCAGGAACAG

6501	GTGGTGGCGG CCCTCGGTGC GCTCGTACTG CTCCACGATG GTGTAGTCCT

6551	CGTTGTGGGA GGTGATGTCC AGCTTGGCGT CCACGTAGTA GTAGCCGGGC

6601	AGCTGCACGG GCTTCTTGGC CATGTAGATG GACTTGAACT CCACCAGGTA

6651	GTGGCCGCCG TCCTTCAGCT TCAGGGCCTT GTGGGTCTCG CCCTTCAGCA

6701	CGCCGTCGCG GGGGTACAGG CGCTCGGTGG AGGCCTCCCA GCCCATGGTC

6751	TTCTTCTGCA TCACGGGGCC GTCGGAGGGG AAGTTCACGC CGATGAACTT

6801	CACCTTGTAG ATGAAGCAGC CGTCCTGCAG GGAGGAGTCC TGGGTCACGG

6851	TCGCCACGCC GCCGTCCTCG AAGTTCATCA CGCGCTCCCA CTTGAAGCCC

6901	TCGGGGAAGG ACAGCTTCTT GTAGTCGGGG ATGTCGGCGG GGTGCTTCAC

6951	GTACACCTTG GAGCCGTACT GGAACTGGGG GGACAGGATG TCCCAGGCGA

7001	AGGGCAGGGG GCCGCCCTTG GTCACCTTCA GCTTCACGGT GTTGTGGCCC

7051	TCGTAGGGGC GGCCCTCGCC CTCGCCCTCG ATCTCGAACT CGTGGCCGTT

7101	CACGGTGCCC TCCATGCGCA CCTTGAAGCG CATGAACTCG GTGATGACGT

7151	TCTCGGAGGA GGCCATTTTG GTAGACTCGA GAGAGATAGA TTTGTAGAGA

7201	GAGACTGGTG ATTTCAGCGT GTCCTCTCCA AATGAAATGA ACTTCCTTAT

7251	ATAGAGGAAG GTCTTGCGAA GGATAGTGGG ATTGTGCGTC ATCCCTTACG

7301	TCAGTGGAGA TATCACATCA ATCCACTTGC TTTGAAGACG TGGTTGGAAC

7351	GTCTTCTTTT TCCACGATGC TCCTCGTGGG TGGGGGTCCA TCTTTGGGAC

7401	CACTGTCGGC AGAGGCATCT TGAACGATAG CCTTTCCTTT ATCGCAATGA

7451	TGGCATTTGT AGGTGCCACC TTCCTTTTCT ACTGTCCTTT TGATGAAGTG

7501	ACAGATAGCT GGGCAATGGA ATCCGAGGAG GTTTCCCGAT ATTACCCTTT

7551	GTTGAAAAGT CTCAATAGCC CTTTGGTCTT CTGAGACTGT ATCTTTGATA

7601	TTCTTGGAGT AGACGAGAGT GTCGTGCTCC ACCATGTTAT CACATCAATC

7651	CACTTGCTTT GAAGACGTGG TTGGAACGTC TTCTTTTTCC ACGATGCTCC

7701	TCGTGGGTGG GGGTCCATCT TTGGGACCAC TGTCGGCAGA GGCATCTTGA

7751	ACGATAGCCT TTCCTTTATC GCAATGATGG CATTTGTAGG TGCCACCTTC

7801	CTTTTCTACT GTCCTTTTGA TGAAGTGACA GATAGCTGGG CAATGGAATC

7851	CGAGGAGGTT TCCCGATATT ACCCTTTGTT GAAAAGTCTC AATAGCCCTT

7901	TGGTCTTCTG AGACTGTATC TTTGATATTC TTGGAGTAGA CGAGAGTGTC

7951	GTGCTCCACC ATGTTGGCAA GCTGCTCTAG CCAATACGCA AACCGCCTCT

8001	CCCCGCGCGT TGGCCGATTC ATTAATGCAG CTGGCACGAC AGGTTTCCCG

8051	ACTGGAAAGC GGGCAGTGAG CGCAACGCAA TTAATGTGAG TTAGCTCACT

8101	CATTAGGCAC CCCAGGCTTT ACACTTTATG CTTCCGGCTC GTATGTTGTG

8151	TGGAATTGTG AGCGGATAAC AATTTCACAC AGGAAACAGC TATGACCATG

8201	ATTACGAATT CAGGTACCAT TTAAATCCTG CAGGGTTTAA ACAGTGTTTT

8251	ACTCCTCATA TTAACTTCGG TCATTAGAGG CCACGATTTG ACACATTTTT

8301	ACTCAAAACA AAATGTTTGC ATATCTCTTA TAATTTCAAA TTCAACACAC

8351	AACAAATAAG AGAAAAAACA AATAATATTA ATTTGAGAAT GAACAAAAGG

8401	ACCATATCAT TCATTAACTC TTCTCCATCC ATTTCCATTT CACAGTTCGA

8451	TAGCGAAAAC CGAATAAAAA ACACAGTAAA TTACAAGCAC AACAAATGGT

8501	ACAAGAAAAA CAGTTTTCCC AATGCCATAA TACTCGAACG GCGCGCCTCA

8551	GCCCATATGC AGGCCGCCGT TGAGCGAGAA GTCGGCGCCG GTCGAGAAAC

8601	CGGACTCCTC CGACGACAAC CAGGCGCAGA TCGAGGCGAT CTCTTCCGGC

8651	AGGCCCAGGC GCTTGACCGG GATCGTCGCG ACGATCTTGT CGAGCACGTC

8701	CTGGCGGATC GCCTTGACCA TGTCGGTGGC GATATAGCCC GGAGAGACCG

8751	TGTTGACGGT CACGCCCTTG GTCGCCACTT CCTGCGCCAG TGCCATGGTG

8801	AAGCCATGCA CGCCGGCCTT GGCGGTGGAG TAGTTGGTCT GGCCGAACTG

8851	GCCCTTCTGC CCGTTCACCG ACGAGATGTT GACGATGCGG CCCCAGCCAC

8901	GGTCGGCCAT GCCGTCGATC ACCTGCTTGG TGACGTTGAA CAGCGAGGTC

8951	AGGTTGGTGT CGATCACCGC ATCCCAGTCG GCGCGGGTCA TCTTGCGGAA

9001	CACCACGTCG CGGGTGATAC CGGCGTTGTT GATCAGCACA TCAACCTCGC

9051	CGACCTCGGA CTTGACCTTG TCGAATGCGG TCTTGGTCGA GTCCCAGTCA

9101	GCCACATTGC CTTCCGAGGC AATGAAATCG AAGCCCAGGG CCTTCTGCTG

9151	CTCCAGCCAC TTTTCGCGGC GCGGCGAGTT GGGGCCGCAA CCGGCCACCA

9201	CACGAAAGCC ATCCTTGGCC AGCCGCTGGC AAATGGCGGT TCCGATACCA

9251	CCCATGCCGC CGGTCACATA CGCAATGCGC TGAGTCACTC TAGAATCTCT

9301	CGTCAATGGT GGCAAATAGG AAAGAGTCTC AAACTTCTTC TTTCCAATTG

9351	GAGGCCACAC CTGCATGCAC TTTACTCTTC CACCATTGCT TGTAATGGAA

9401	GTAATGTCAG TGTTGACCTT CTTCACTGGG AATCCAGTCA TGGATTTGAG

9451	GCCGCCGAAT GGAGCCACTG CGGCGGATTG CCCCCTAGAG GCACGGCTGA

9501	CTGTTGTCAC AGCGGAAGAG GATATCATAG AAGCCATTTT ACTAGTAAGA

9551	AGCTGAAAAT ATCAAAAGAA GGAACAGTCA TTAATCTATT GCATGTACTA

9601	GATTTTAGAT ATGAGTGGTC AAAAAAAACT TACGTTAATA ACGATGAAGA

9651	AGACAATGAT CCTCAGCACA ATCTCTCTCT CTCTCTCTTG GCTTCTCTTC

9701	TGGTGAATAG CACGAGAGAG GGTTTAAATG GAAGGCTCGT GGGTCCAAAA

9751	TGGGTGGCGG AGGAAATAGG AGAAGTAGGC AGTGACAAGT AATGTAGTAT

9801	TTAGTATTTG ATGAATGACA CATTTTCATT TCAGCATCAT CACCAACCAT

9851	CCTTTTGTTC CTTTGCTTCA ACTGTCACTT TCAATTGACA AAATTTTTTA

9901	TGTTTTCATG AGAAAACTAA ATTCTTATAA AGATTCATCT TCTTGAGTAT

9951	TATACGTGTA GTTTATGAAC AACACGTGTT GTTCCTATAT TTTTGTTCTG

10001	TTACCTCTAG AATAAAGTTG TCACCATTTC ATGAGTTCAA TTTTTCTTTA

10051	ATAGCCCCAA AAACAAAAGA TGATTCACAA GAAAGATGCG AATATTTTGC

10101	TATGAATCTT TTCTTAAGAG AAGCAATTAC ATTTTCACAA TAAAATTAGA

10151	TCCACGACTT AACCTAGTTT ATGTTGATTA TTTCTAGTGT TAGTATTAAG

10201	CAAAAATAAA ACTTATGAAT ACGAAGGCCT TTAAAGGAAA CTAAAGAAAG

10251	GACAAGGTAT AAACGTCCTA GAAAGTTCTA GGGTTTAGGC TTAGGGTCTA

10301	AGATATATGC TTTGAGTTTT ATGGCTTAGT AACACATTTT TGTAACACTT

10351	CTTTGTAACA TTTCTTGATA TGTTGGAGAA GTAACTCGTC TGGACAATAG

10401	TTATTTCCAA TATATAGGAA AAACGGCCTA AACAATAGCC GACGGGGACA

10451	AATACATCAT AAACAAAAAA TCCCGGTTAC AAACTTCCTA AAAAGCCATT

10501	CGGTCCACTC CGTTAAGCCT GAACTGTGCC TCCGTTATGC AAAAACGCCG

10551	TTGACCATCC GTAACCTAGT TGACTGACGG ATTATGGATT TAATCCGTTT

10601	TAAGGCCGTT AATAACACCA AAACGACGTC GTTTTGGTGT TTAAATTTTT

10651	TTTAACAACA ATTAAACCAA ACGACGTCGT TTTGGTTTAA TTAAATTTTT

10701	TTATCAAAAA CCCAAGCCCA AGCCCAAAAC TCTTAACAAA AGATAAAGCC

10751	CATCTCTATT TTTTCTAATT AAAACGCACA GCATTATGTT TCTTCTCTAA

10801	CGGATATATT TTCAATCTCA TAAATTGGGG ATTAGGGTTC TTATTTCCCA

10851	ATTCTCAATC TCTCAAAATT CTCCAAAATT CTCTGAAATT GATAATGCCT

10901	TCTTCTTCTT CAAACTCGTT TTTCTCTTTT GACAGTGAGC TTGAAGATGA

10951	TAACCATCGT GGTTTTCCTA AGACCTGTCG ATTTGGATGT CGTGTTGTGA

11001	TCAGAACCTC AAGAACTCCA AAAAACCTAG GTAGATTATT CCATACCTGT

11051	GAGAAAAATT TCAAAAGAGG AGGATTCCAC ACCTGGAAGT GGACTGATGT

11101	GTCTTTAGTA GAAGAAGTAG AGGACATAAA GGCTTACATT CATAACCGTG

11151	AGAAGTGTCA CGATGAAGAA ATGTTATTAT TGAAGGCTCA GATTCGTGGC

11201	TGTGAGAAGA TGATTGAAGG CTTGAAAGGA GAAGCAAAAC GTATGAAGCT

11251	AATTGTTGTT GCCGGAATAG TTGTGTTTGG TTGCTTTTTG TGTCTCTCTA

11301	AGTGATGTAT GAGATGAATG TTTGTGTATG TGATGTTGTT TTGTCTCAAT

11351	AATTAGTCAC TGATGTTGTA TGTAATGTTG TGTTTTGCAT CTCTAATTAG

11401	TTAATAATGA ATGTTGTTCT TATGTAATGT TTGATTTAAT CAATGGCTTT

11451	TGCAAATAAA TCCATAACAG AACNTATTCA ATATTTTCGA AAACATAACA

11501	AAGGTTTCAA AAGAAATTGC ATGTTGATTA GCTGAGTTTT CAAACAAAAT

11551	GCATTACATA GACAGACCCT GCTTCATAAT CCCCAAAACA CAAAAGAGAA

11601	GCATGCTAAT AACCGCAACT AATATCCAAA GACAGCTTCA TAATCCCAAA

11651	ACACAAAAAA AGAAGATTCA TAACCGATCC TTCATGTATT TAAAGAAAAT

11701	CAGACAACAA GCAAAGACTT AATCTTCCTG AGTAACTGAT GAGCTCAAGT

11751	CGACGTTTAA ACAGTGTTTT ACTCCTCATA TTAACTTCGG TCATTAGAGG

11801	CCACGATTTG ACACATTTTT ACTCAAAACA AAATGTTTGC ATATCTCTTA

11851	TAATTTCAAA TTCAACACAC AACAAATAAG AGAAAAAACA AATAATATTA

11901	ATTTGAGAAT GAACAAAAGG ACCATATCAT TCATTAACTC TTCTCCATCC

11951	ATTTCCATTT CACAGTTCGA TAGCGAAAAC CGAATAAAAA ACACAGTAAA

12001	TTACAAGCAC AACAAATGGT ACAAGAAAAA CAGTTTTCCC AATGCCATAA

12051	TACTCGAACT ACGTATTATT TGCGCTCGAC TGCCAGCGCC ACGCCCATGC

12101	CGCCGCCGAT GCACAGCGAG GCCAGGCCCT TCTTCGCGTC ACGGCGCTTC

12151	ATCTCGTGCA GCAGCGTCAC CAGGATACGG CAGCCCGACG CGCCGATCGG

12201	GTGGCCGATG GCGATGGCGC CGCCGTTCAC ATTGACCTTG GAGGTGTCCC

12251	AGCCCATCTG CTGGTGCACC GCCAGCGCCT GCGCGGCAAA GGCCTCGTTG

12301	ATCTCCATCA GGTCCAGGTC TTGCGGGGTC CACTCGGCGC GCGACAGGGC

12351	GCGCTTGGAG GCCGGCACCG GGCCCATGCC CATCACCTTG GGATCGACAC

12401	CGGCGTTGGC ATAGCTCTTG ATCGTGGCCA GCGGGGTCAG GCCCAGTTCC

12451	TTGGCCTTGG CCGCCGACAT CACCACCACC GCGGCGGCGC CGTCGTTCAG

12501	GCCCGAGGCG TTGGCCGCGG TCACCGTGCC GGCCTTGTCG AAGGCGGGCT

12551	TGAGGCCGGA CATGCTGTCC AGCGTGGCGC CCTGGCGCAC GAACTCGTCG

12601	GTCTTGAAGG CCACCGGGTC GCCCTTGCGC TGCGGGATCA GCACCGGGAC

12651	GATCTCTTCG TCAAACTTGC CGGCCTTCTG CGCGGCTTCG GCCTTGTTCT

12701	GCGAGCCGAC GGCGAACTCA TCCTGCGCCT CGCGTGTGAT GCCGTATTCC

12751	TTGGCCACGT TCTCGGCGGT GATGCCCATG TGGTACTGGT TGTACACGTC

12801	CCACAGGCCG TCGACGATCA TGGTGTCGAC CAGCTTGGCA TCGCCCATGC

12851	GGAAACCATC GCGCGAGCCC GGCAGCACGT GCGGGGCGGC GCTCATGTTT

12901	TCCTGGCCGC CGGCCACCAC GATCTCGGCG TCGCCCGCCA TGATCGCGTT

12951	GGCGGCCAGC ATCACGGCCT TCAGGCCCGA GCCGCACACC TTGTTGATGG

13001	TCATGGCCGG CACCATCGCC GGCAGGCCGG CCTTGATCGC GGCCTGGCGT

13051	GCGGGGTTCT GGCCCGAACC GGCGGTCAGC ACCTGGCCCA TGATGACTTC

13101	GCTCACCTGC TCCGGCTTGA CGCCGGCGCG CGCCGGCGCG GCCTTGATGA

13151	CCACGGCACC CAGTTCCGGT GCGGGGTTCT TGGCCAGCGA GCCGCCAAAC

13201	TTGCCGACCG CGGTGCGGGC GGCGGATACG ATGACAACGT CAGTCACTCT

13251	AGAATCTCTC GTCAATGGTG GCAAATAGGA AAGAGTCTCA AACTTCTTCT

13301	TTCCAATTGG AGGCCACACC TGCATGCACT TTACTCTTCC ACCATTGCTT

13351	GTAATGGAAG TAATGTCAGT GTTGACCTTC TTCACTGGGA ATCCAGTCAT

13401	GGATTTGAGG CCGCCGAATG GAGCCACTGC GGCGGATTGC CCCCTAGAGG

13451	CACGGCTGAC TGTTGTCACA GCCGAACAGG ATATCATAGA AGCCATTTTG

13501	GATCCAAGAA GCTGAAAATA TCAAAAGAAG GAACAGTCAT TAATCTATTG

13551	CATGTACTAG ATTTTAGATA TGAGTGGTCA AAAAAAACTT ACGTTAATAA

13601	CGATGAAGAA GACAATGATC CTCAGCACAA TCTCTCTCTC TCTCTCTTGG

13651	CTTCTCTTCT GGTGAATAGC ACGAGAGAGG GTTTAAATGG AAGGCTCGTG

13701	GGTCCAAAAT GGGTGGCGGA GGAAATAGGA GAAGTAGGCA GTGACAAGTA

13751	ATGTAGTATT TAGTATTTGA TGAATGACAC ATTTTCATTT CAGCATCATC

13801	ACCAACCATC CTTTTGTTCC TTTGCTTCAA CTGTCACTTT CAATTGACAA

13851	AATTTTTTAT GTTTTCATGA GAAAACTAAA TTCTTATAAA GATTCATCTT

13901	CTTGAGTATT ATACGTGTAG TTTATGAACA ACACGTGTTG TTCCTATATT

13951	TTTGTTCTGT TACCTCTAGA ATAAAGTTGT CACCATTTCA TGAGTTCAAT

14001	TTTTCTTTAA TAGCCCCAAA AACAAAAGAT GATTCACAAG AAAGATGCGA

14051	ATATTTTGCT ATGAATCTTT TCTTAAGAGA AGCAATTACA TTTTCACAAT

14101	AAAATTAGAT CCACGACTTA ACCTAGTTTA TGTTGATTAT TTCTAGTGTT

14151	AGTATTAAGC ACAAATAAGA CTTATGAATA CGAAGGCCTT TAAAGGAAAC

14201	TAAAGAAAGG ACAAGGTATA AACGTCCTAG AAAGTTCTAG GGTTTAGGCT

14251	TAGGGTCTAA GATATATGCT TTGAGTTTTA TGGCTTAGTA ACACATTTTT

14301	GTAACACTTC TTTGTAACAT TTCTTGATAT GTTGGAGAAG TAACTCGTCT

14351	GGACAATAGT TATTTCCAAT ATATAGGAAA AACTTCCTAA ACAATAGCCG

14401	ACGGGGACAA ATACATCATA AACAAAAGAT CCCGGTTACA AACTTCCTAA

14451	AAAGCCATTC GGTCCACTCC GTTAAGCCTG AACTGTGCCT CCGTTATGCA

14501	AAAACGCCGT TGACCATCCG TAACCTAGTT GACTGACGGA TTATGGATTT

14551	AATCCGTTTT AAGGCCGTTA ATAACACCAA AACGACGTCG TTTTGGTGTT

14601	TTAATTTTTT TTAACAACAA TTAAACCAAA CGACGTCGTT TTGGTTTAAT

14651	TAAATTTTTT TATCAAAAAC CCAAGCCCAA GCCCAAAACT CTTAACAAAA

14701	GATAAAGCCC ATCTCTATTT TTTCTAATTA AAACGCACAG CATTATGTTT

14751	CTTCTCTAAC GGATATATTT TCAATCTCAT AAATTGGGGA TTAGGGTTCT

14801	TATTTCCCAA TTCTCAATCT CTCAAAATTC TCCAAAATTC TCTGAAATTG

14851	ATAATGCCTT CTTCTTCTTC AAACTCGTTT TTCTCTTTTG ACAGTGAGCT

14901	TGAAGATGAT AACCATCGTG GTTTTCCTAA GACCTGTCGA TTTGGATGTC

14951	GTGTTGTGAT CAGAACCTCA ATAACACCAA AAAACCTAGG TAGATTATTC

15001	CATACCTGTG AGAAAAATTT CAAAAGAGGA GGATTCCACA CCTGGAAGTG

15051	GACTGATGTG TCTTTAGTAG AAGAAGTAGA GGACATAAAG GCTTACATTC

15101	ATAACCGTGA GAAGTGTCAC GATGAAGAAA TGTTATTATT GAAGGCTCAG

15151	ATTCGTGGCT GTGAGAAGAT GATTGAAGGC TTGAAAGGAG AAGCAAAACG

15201	TATGAAGCTA ATTGTTCTTG CCGGAATAGT TGTGTTTGGT TGCTTTTTGT

15251	GTCTCTCTAA GTGATGTATG AGATGAATGT TTGTGTATGT GATGTTGTTT

15301	TGTCTCAATA ATTAGTCACT GATGTTGTAT GTAATGTTGT GTTTTGCATC

15351	TCTAATTAGT TAATAATGAA TGTTGTTCTT ATGTAATGTT TGATTTAATC

15401	AATGGCTTTT GCAAATAAAT CCATAACAGA ACNTATTCAA TATTTTCGAA

15451	AACATAACAA AGGTTTCAAA AGAAATTGCA TTAGCATTAG CTGAGTTTTC

15501	AAACAAAATG CATTACATAG ACAGACCCTG CTTCATAATC CCCAAAACAC

15551	AAAAGAGAAG CATGCTAATA ACCGCAACTA ATATCCAAAG ACAGCTTCAT

15601	AATCCCAAAA CACAAAAAAA GAAGATTCAT AACCGATCCT TCATGTATTT

15651	AAAGAAAATC AGACAACAAG CAAAGACTTA ATCTTCCTGA GTAATGGAAG

15701	AGCTCAACTG CAGGTTTAAA CAGTGTTTTA CTCCTCATAT TAACTTCGGT

15751	CATTAGAGGC CACGATTTGA CACATTTTTA CTCAAAACAA AATGTTTGCA

15801	TATCTCTTAT AATTTCAAAT TCAACACACA ACAAATAAGA GAAAAAACAA

15851	ATAATATTAA TTTGAGAATG AACAAAAGGA CCATATCATT CATTAACTCT

15901	TCTCCATCCA TTTCCATTTC ACAGTTCGAT AGCGAAAACC GAATAAAAAA

15951	CACAGTAAAT TACAAGCACA ACAAATGGTA CAAGAAAAAC AGTTTTCCCA

16001	ATGCCATAAT ACTCGAACGC GATCGCTCAG CCCTTGGCTT TGACGTAACG

16051	GCCGGGCGCC GCCTCGATCG CGGTGTAGCG GGCGTTGCCG GGCTTGGCCT

16101	TGGGCTTGAC CTTCTTGCCG CCATGCTGGG TCAGGAACCC GGCCCATTGC

16151	GGCCACCAGC TGCCCGGCAC TTCCTGCGCG CCATCGAACC AGGCCTGGGC

16201	ATCGGCGGCG CCACCGTCGT TGATCCAGTA GCTGCGCTTG TTCTTGGCCA

16251	CCGAGTTGAT CACGCCGGCG ATATGGCCGG ACGCGCCCAG CACGAAGCGG

16301	TTGGCGCCCG GCTTGCCCTG GTTGAGGATG TCGAGCGAAC CGTACGCCGA

16351	CATCCACGGC ACGATGTGGT CTTCGCGCGA ACCGTAGATG AAGGCCGGGG

16401	CGTCGATCAG GCCGAGGTCG ATCTTTTCGC CGGCCACCGT CAGCTTGCCC

16451	GGCACTTTCA GGCTGTTTTC CAGGTAGGTG TTGCGCAGGT ACCAGCAGAA

16501	CATCGGGCCC GGCAAATTGG TGCTGTCCGA ATTCCAGAAC AGCAGGTCAA

16551	ACGCCGCCGG CTCATTGCCT TTGAGGTAGT TCGACTGCAC ATAGTTCCAT

16601	ACCAGGTCGT TCGGACGCAG GCTCGAGAAG GTCGAGGCCA GGTCACGGCC

16651	CGGCATCAGG CCGCCATCGC GCAATTGCTG TTCACGCAGC GCGACCTGGG

16701	TTTCATCGAC GAAGACGTCG AGCACGCCGG TGTCGCTGAA GTCGAGGAAG

16751	GTGGTCAGCA GGGTCAGGCT GGCCGCCGGG TGCTGGCCAC GCGCCGCCAG

16801	TACCGCCAGT GCGGTGGCAA CGATGGTGCC GCCCACGCAG AAGCCGAACA

16851	TGTTCAGCTT GTCCTGGCCG CTGACGTCCT GGACGATGCG GATCGCTTCG

16901	ATCACGCCCT GCTCCACGTA GTCGTCCCAG GTGGTGCCGG CCAGCGACTT

16951	GTCCGGATTG CTCCACGAGA TCAGGAACAC GGTGTTGCCC TGCTCCACCG

17001	CGTAGCGCAC CAGCGAATTT TCCGGTTGCA GGTCGAGGAT GTAGAACTTG

17051	TTGATGCACG GCGGCACCAT CAACAGCGGG CGCTGGCTGA CCGTCGGCGT

17101	GGTCGGCGTG TACTGGATCA GCTGGAACAG CGGATTTTCG TAAATCACGG

17151	TGCCCGGGGT AATGGCCAGG TTGCGGCCCA CTTCAAAGGC CGATTCGTCC

17201	GACAGCGAGA TATGGCCCTT GTTGATATCG CCCAGCATAT TGACCAGGCC

17251	ACGCGTCAGG CTCTCGCCCT TGGTTTCAAT CAGTTTTTGC TGCGCTTCCG

17301	GGTTGGTGGC GAGGAAGTTC GCGGGCGACA TGGCATCAAT CACCTGCTGC

17351	ACGGCAAAGC GTATTTTCTG CTTTTGCTGG GGTGCGGTGT CCACCGCCTC

17401	CACCATGGCA CTGAGGAATT TGGCGTTGAG CAGGTAAGAT GCGGCATTGA

17451	AGGCCGACAT CGGATTGCCC TGCCAGGCTG CCGAGCTGAA GCGGCGGTCG

17501	CTGACGGCTG GCGCCTTGCC AGCCAAAAAA TCCTGCCACA ACGCGGTGAA

17551	GTCACGCAGA TAATCGTTTT TCAGCTGCTC CATCGCTTCC GGTTTGAGCG

17601	CAACGCCGAT ATCCTGCAAC ATGGTGGCCA TCGGGTTCGC CTCGGTGGTG

17651	GGCGCCTTGC TGAACCAGGA TTGCCACTGC AGCTCATCGT TGTTCTTGTT

17701	ACTCACTCTA GAATCTCTCG TCAATGGTGG CAAATAGGAA AGAGTCTCAA

17751	ACTTCTTCTT TCCAATTGGA GGCCACACCT GCATGCACTT TACTCTTCCA

17801	CCATTGCTTG TAATGGAAGT AATGTCAGTG TTGACCTTCT TCACTGGGAA

17851	TCCAGTCATG GATTTGAGGC CGCCGAATGG AGCCACTGCG GCGGATTGCC

17901	CCCTAGAGGC ACGGCTGACT GTTGTCACAG CGGAAGAGGA TATCATAGAA

17951	GCCATTTTTG TACAAAGAAG CTGAAAATAT CAAAAGAAGG AACAGTCATT

18001	AATCTATTGC ATGTACTAGA TTTTAGATAT GAGTGGTCAA AAAAAACTTA

18051	CGTTAATAAC GATGAAGAAG ACAATGATCC TCAGCACAAT CTCTCTCTCT

18101	CTCTCTTGGC TTCTCTTCTG GTGAATAGCA CGAGAGAGGG TTTAAATGGA

18151	AGGCTCGTGG GTCCAAAATG GGTGGCGGAG GAAATAGGAG AAGTAGGCAG

18201	TGACAAGTAA TGTAGTATTT AGTATTTGAT GAATGACACA TTTTCATTTC

18251	AGCATCATCA CCAACCATCC TTTTGTTCCT TTGCTTCAAC TGTCACTTTC

18301	AATTGACAAA ATTTTTTATG TTTTCATGAG AAAACTAAAT TCTTATAAAG

18351	ATTCATCTTC TTGAGTATTA TACGTGTAGT TTATGAACAA CACGTGTTGT

18401	TCCTATATTT TTGTTCTGTT ACCTCTAGAA TAAAGTTGTC ACCATTTCAT

18451	GAGTTCAATT TTTCTTTAAT AGCCCCAAAA ACAAAAGATG ATTCACAAGA

18501	AAGATGCGAA TATTTTGCTA TGAATCTTTT CTTAAGAGAA GCAATTACAT

18551	TTTCACAATA AAATTAGATC CACGACTTAA CCTAGTTTAT GTTGATTATT

18601	TCTAGTGTTA GTATTAAGCA AAAATAAAAC TTATGAATAC GAAGGCCTTT

18651	AAAGGAAACT AAAGAAAGGA CAAGGTATAA ACGTCCTAGA AAGTTCTAGG

18701	GTTTAGGCTT AGGGTCTAAG ATATATGCTT TGAGTTTTAT GGCTTAGTAA

18751	CACATTTTTG TAACACTTCT TTGTAACATT TCTTGATATG TTGGAGAAGT

18801	AACTCGTCTG GACAATAGTT ATTTCCAATA TATAGGAAAA ACGGCCTAAA

18851	CAATAGCCGA CGGGGACAAA TACATCATAA ACAAAAAATC CCGGTTACAA

18901	ACTTCCTAAA AAGCCATTCG GTCCACTCCG TTAAGCCTGA ACTGTGCCTC

18951	CGTTATGCAA AAACGCCGTT GACCATCCGT AACCTAGTTG ACTGACGGAT

19001	TATGGATTTA ATCCGTTTTA AGGCCGTTAA TAACACCAAA ACGACGTCGT

19051	TTTGGTGTTT TAATTTTTTT TAACAACAAT TAAACCAAAC GACGTCGTTT

19101	TGGTTTAATT AAATTTTTTT ATCAAAAACC CAAGCCCAAG CCCAAAACTC

19151	TTAACAAAAG ATAAAGCCCA TCTCTATTTT TTCTAATTAA AACGCACAGC

19201	ATTATGTTTC TTCTCTAACG GATATATTTT CAATCTCATA AATTGGGGAT

19251	TAGGGTTCTT ATTTCCCAAT TCTCAATCTC TAACACTTCT CCAAAATTCT

19301	CTGAAATTGA TAATGCCTTC TTCTTCTTCA AACTCGTTTT TCTCTTTTGA

19351	CAGTGAGCTT GAAGATGATA ACCATCGTGG TTTTCCTAAG ACCTGTCGAT

19401	TTGGATGTCG TGTTGTGATC AGAACCTCAA GAACTCCAAA AAACCTAGGT

19451	AGATTATTCC ATACCTGTGA GAAAAATTTC AAAAGAGGAG GATTCCACAC

19501	CTGGAAGTGG ACTGATGTGT CTTTAGTAGA AGAAGTAGAG GACATAAAGG

19551	CTTACATTCA TAACCGTGAG AAGTGTCACG ATGAAGAAAT GTTATTATTG

19601	AAGGCTCAGA TTCGTGGCTG TGAGAAGATG ATTGAAGGCT TGAAAGGAGA

19651	AGCAAAACGT ATGAAGCTAA TTGTTGTTGC CGGAATAGTT GTGTTTGGTT

19701	GCTTTTTGTG TCTCTCTAAG TGATGTATGA GATGAATGTT TGTGTATGTG

19751	ATGTTGTTTT GTCTCAATAA TTAGTCACTG ATGTTGTATG TAATGTTGTG

19801	TTTTGCATCT CTAATTAGTT AATAATGAAT GTTGTTCTTA TGTAATGTTT

19851	GATTTAATCA ATGGCTTTTG CAAATAAATC CATAACAGAA CNTATTCAAT

19901	ATTTTCGAAA ACATAACAAA GGTTTCAAAA GAAATTGCAT TAGCATTAGC

19951	TGAGTTTTCA AACAAAATGC ATTACATAGA CAGACCCTGC TTCATAATCC

20001	CCAAAACACA AAAGAGAAGC ATGCTAATAA CCGCAACTAA TATCCAAAGA

20051	CAGCTTCATA ATCCCAAAAC ACAAAAAAAG AAGATTCATA ACCGATCCTT

20101	CATGTATTTA AAGAAAATCA GACAACAAGC AAAGACTTAA TCTTCCTGAG

20151	TAACTGATGA GCTCAAAAGC TTGGCACTGG CCGTCGTTTT ACAACGTCGT

20201	GACTGGGAAA ACCCTGGCGT TACCCAACTT AATCGCCTTG CAGCACATCC

20251	CCCTTTCGCC AGCTGGCGTA ATAGCGAAGA GGCCCGCACC GATCGCCCTT

20301	CCCAACAGTT GCGCAGCCTG AATGGCGAAT GCTAGAGCAG CTTGAGCTTG

20351	GATCAGATTG TCGTTTCCCG CCTTCAGTTT AAACTATCAG TGTTTGACAG

20401	GATATATTGG CGGGTAAACC TAAGAGAAAA GAGCGTTTAT TAGAATAACG

20451	GATATTTAAA AGGGCGTGAA AAGGTTTATC CGTTCGTCCA TTTGTATGTG

Claims

1. An oilseed comprising greater than 7% polyhydroxyalkanoate (PHA) dry weight of the oilseed, wherein germination of the oilseed is impaired relative to an oilseed having less than 7% polyhydroxyalkanoate.

2. The oilseed of claim 1, wherein the PHA comprises (poly) 3-hydroxybutyrate (PHB).

3. The oilseed of claim 1 comprises greater than 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% or 19% of the dry weight of the seed.

4. The oilseed of claim 1, wherein the oilseed is produced by a transgenic plant genetically engineered to produce PHA.

5. The oilseed of claim 4, wherein the PHA is PHB.

6. The oilseed of claim 4, wherein the plant transformed to produce the transgenic plant is selected from the group consisting of members of the Brassica family: B. napus, B. rapa, B. carinata and B. juncea; industrial oilseeds: Camelina sativa, Crambe, jatropha, castor; Arabidopsis thaliana; Calendula, Cuphea; maize; soybean; cottonseed; sunflower; palm; coconut; safflower; peanut; mustards including Sinapis alba; and tobacco.

7. The oilseed of claim 1, wherein germination of the oilseed is impaired by 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% relative to an oilseed comprising less than 7% PHA.

8. A transgenic plant genetically engineered to produce PHA, wherein the transgenic plant produces the oilseed of claim 1.

9. The transgenic plant of claim 8 wherein the plant transformed to produce the oilseed of claim 1 is selected from the group consisting of members of the Brassica family; B. napus, B. rapa, B. carinata and B. juncea; industrial oilseeds: Camelina sativa, Crambe, Jatropha, castor; Arabidopsis thaliana; Calendula, Cuphea; maize; soybean; cottonseed; sunflower; palm; coconut; safflower; peanut; mustards: Sinapis alba; and tobacco.

10. (canceled)

11. A method for producing a hybrid transgenic plant line comprising crossing a plant line comprising one or more PHB biosynthetic pathway genes with a plant line containing the remaining PHB biosynthetic pathway gene(s) needed to complete the PHB biosynthetic pathway.

12. The method of claim 11 wherein the plant lines comprise cytoplasmic male sterility (CMS) controlled by an extranuclear genome.

13. The method of claim 11 wherein the male sterile line is maintained by crossing with a maintainer line that is genetically identical and comprises normal fertile cytoplasm.

14. The method of claim 13 wherein the maintainer line is transformed with one or more genes for the PHB biosynthetic pathway.

15. The method of claim 14 wherein crossing the transformed maintainer line with the original male sterile line produces a male sterile line possessing a portion of the PHB biosynthetic pathway.

16. The method of claim 15 wherein insertion of the phaA and phaC genes into the maintainer line and crossing with the original male cytoplasmic sterile line forms a male sterile line containing the phaA and phaC genes.