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WO2006031721A2 - Genie genetique utilise pour parvenir a la sterilite male de plantes - Google Patents

Genie genetique utilise pour parvenir a la sterilite male de plantes Download PDF

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WO2006031721A2
WO2006031721A2 PCT/US2005/032402 US2005032402W WO2006031721A2 WO 2006031721 A2 WO2006031721 A2 WO 2006031721A2 US 2005032402 W US2005032402 W US 2005032402W WO 2006031721 A2 WO2006031721 A2 WO 2006031721A2
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plant
plastid
stably transformed
gene
sequence
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PCT/US2005/032402
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WO2006031721A3 (fr
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Henry Daniell
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University Of Central Florida Research Foundation, Inc.
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Priority to US11/575,090 priority Critical patent/US20090007294A1/en
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Publication of WO2006031721A3 publication Critical patent/WO2006031721A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8214—Plastid transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility

Definitions

  • male-fertile parental and male-sterile transformed lines would be developed from the same inbred, they should be highly uniform and possess the same nuclear genotype, excluding mutations and residual heterozygosity (4). Therefore, the male-fertile parental line becomes the maintainer line to seed- propagate the newly transformed male-sterile line (4). A few generations of seed increases would produce a CMS-maintainer pair for hybrid seed production.
  • organellar transformation would be the diversification of CMS sources used in commercial hybrid-seed production. Transformation of the chloroplast genome would allow breeders to introduce different male-sterility- inducing factors into superior inbred lines. Introduction of a male-sterility inducing transgene into one of the organellar genomes of a higher plant would be a major breakthrough in the production of male- sterile inbred lines (4). This technique would be of great potential importance in the production of hybrid crops by avoiding generations of backcrossing, an approach especially advantageous for crop plants with longer generation times (4). Moreover, transgenes that are engineered into our annual crops could be introgressed into wild crops, persist in the environment and have negative ecological consequences may be necessary to engineer a male sterility system that is 100% effective (18).
  • PHB synthesis takes place by the consecutive metabolic action of ⁇ -ketothiolase (phaA gene), acetoacetyl-CoA reductase (phaB) and PHB synthase (phaC).
  • phaA gene ⁇ -ketothiolase
  • phaB acetoacetyl-CoA reductase
  • phaC PHB synthase
  • Nawrath et al (20) introduced the phbA, phbB and phbC genes in individual nuclear Arabidopsis transgenic lines and reconstructed the entire pathway, targeting all enzymes to the plastids.
  • This approach resulted in PHB expression up to 14% leaf dry weight, and no pleiotropic effects.
  • the subject invention is directed to engineered male sterile phenotype in plants.
  • the subject invention is based on the inventors' discovery that transformation of the ⁇ -ketothiolase (phaA gene) into the chloroplast genome, and its expression, enables the regeneration of transgenic plants that have overcome pleiotropic effects such as stunted phenotype and chlorosis observed during polyhydroxybutyrate expression (21, 23) but maintained complete (100%) male sterility.
  • FIG. 1 Molecular characterization of transgenic lines.
  • A Schematic representation of the transformed chloroplast genome and the pLDR-5'UTR-p/z ⁇ A cassette. Annealing sites for the primer pairs and expected sizes of PCR products are shown. Bam ⁇ I restriction sites, DNA fragment produced after restriction digestion and the phaA probe, used in Southern-blot analyses are shown.
  • B the map shows the wild-type chloroplast genome, restriction digestion sites used for Southern-blot analysis, expected fragment size after digestion and probing with the flanking sequence.
  • FIG. 2 A PCR analysis of wild type and putative transformants.
  • Top panel transgenic line 4B; bottom panel is transgenic line 4A.
  • A 1: Untransformed wild type; 2: 3P-3M (1.65 kb); 3: 4P- 4M (1.65 kb); 4: 5P-2M (3.56 kb); 5: 5P-3'phaA (2.0 kb); 6: 5V-phaA internal (1.5 kb); 7: positive control (pLD-5'V ⁇ R-phbA plasmid DNA) 5V-phbA internal (1.5 kb); M: marker.
  • B and D Southern-blot analysis of T 0 and T 1 generation transgenic lines, respectively, with the chloroplast flanking sequence probe; 10 kb fragment shows integration of transgenes, 7.1 kb fragment shows wild type fragment.
  • C and E transgenic T 0 and Ti plants, respectively, and wild type plant probed with the phaA probe. 10 kb fragment observed in transgenic lines but not in the wild type.
  • F and G northern-blot analysis using the phaA and aadA probes, respectively.
  • Mononocistron (a: 5'UTR-£>/z ⁇ A; 1,384 nt) and polycistrons (b: aadA-phaA, 2,255 nt; c: from native 16S Vrrn, 4,723 nt) containing the phaA gene are observed in the transgenic lines when the phaA is used. Only polycistrons are observed with the aadA probe.
  • B to G 1 , untransformed plant; 2 to 4, chloroplast transgenic lines 4A, 4B, and 4C, respectively; B, blank lane.
  • FIG. 3 ⁇ -ketothiolase characterization in transgenic lines.
  • A coomassie-stained gel shows abundant ⁇ -ketothiolase expression in transgenic lines; 15 ⁇ g total plant protein was loaded per lane (a: Dl protein; b: ⁇ -ketothiolase, 40.8 kDa; c: RBCL).
  • M marker; 1-3: transgenic lines 4A, 4B, 4C, respectively; 4: 4A Ti generation; 5: wild type; 6: bacterial purified ⁇ -ketothiolase.
  • B western-blot analysis: 10 ⁇ g total plant protein from transgenic lines and wild type was loaded per lane; anti- ⁇ - ketothiolase antibody was used.
  • transgenic lines 40.8 kDa monomers (a), dimer (b), trimer (c) and tetramer (d) can be observed in transgenic lines.
  • 1 untransformed plant; 2-4: transgenic lines 4A, 4B, 4C, respectively; 5-6: 4A and 4B Ti generation, respectively.
  • A-C Flowers from transgenic lines; note the absence of fruit capsules and fallen flowers.
  • D and E wild type tobacco flowers and fruit capsules.
  • F and G comparison of stamens and stigma. Note shorter stamens in transgenic lines (F) compared to untransformed (G).
  • H and I comparison of mature anthers. Note abundant pollen in untransformed plant (I) and the lack of pollen in transformed anther (H).
  • J transgenic fruit capsule with seeds developed after pollination of transgenic stigma with untransformed pollen.
  • K germination and growth of Ti seedlings on MS medium with 500 ⁇ g/ml spectinomycin.
  • FIG. 5 Comparison of growth and development.
  • A Untransformed (WT) and To generation transgenic (T) line (4A) grown for two months in soil.
  • B Untransformed plant (WT) and Ti generation independent transgenic lines 4A, 4B, 4C and 4D 1.5 month after germination.
  • FIG. 6 SEM pictures of pollen grains in anthers of wild type (A-C) and transgenic (D-F) plants at different magnifications.
  • FIG. 7 ⁇ -Ketothiolase Expression in Anthers.
  • A pigmentation of anthers during flower development in transgenic plants, Stage 1 of flower development is shown.
  • B northern-blot analysis of flower and anthers. Monocistrons and polycistron are observed. 3 ⁇ g of total plant RNA was loaded per lane and thephaA probe was used.
  • M marker; 1 : transgenic flower; 2-3: transgenic anthers; 4: wild type flower; 5: transgenic leaf; 6: wild type leaf.
  • C ⁇ -ketothiolase expression in transgenic flower and anther detected by western-blot analysis, lanes 2 and 3, respectively.
  • RNA and protein samples used per lane were the product of the combined extraction from flowers or anthers from stages 1 and 3.
  • Fig. 8 Analysis of anther development. Bright-field photographs of untransformed (wt) and transgenic anthers at different developmental stages (S). Anthers at the designated stages were fixed, embedded with paraffin, and sliced into 5 and 10 ⁇ m transverse sections. The fixed sections were stained with toluidine blue and visualized under the light microscope at a magnifications of xlOO.
  • C connective tissue
  • E epidermis
  • En endothecium
  • MMC microspore mother cells
  • Msp microspores
  • PS pollen sac
  • S stomium
  • T tapetum.
  • Fig. 9 Reversibility of male fertility after 10 days under continuous illumination.
  • A transgenic flower after 9 days in continuous light. A, note normal length of the anther filaments and pollen grains. B, fully developed fruit capsules containing seeds from transgenic lines. C, abundant seeds from transgenic fruit capsule. D, seedlings produced via the reversibility to male fertility. Transgenic seeds germinated in MS medium supplied with 500 ⁇ g/mL spectinomycin. E, bleached wild type tobacco seedlings.
  • Fig. 10 shows the nucleic acid sequence of the phaA gene.
  • a polynucleotide sequence is "homologous" with the polynucleotide sequence according to the invention (Acinetobacter sp. ⁇ -ketothiolase gene, also referred to asphaA gene; accession no: L37761, see also Fig.
  • nucleic acid sequences corresponds to the sequence according to the invention; wherein corresponds refers to the percent identity of the nucleic acid sequences; or percent identity of the amino acid sequences that the nucleic acid sequences encode to the amino acid sequences described herein, as determined by algorithms commonly employed by those skilled in this art; or those sequences that are characterized by their ability to hybridize to a sequence according to the invention (as described below).
  • percent identity is calculated by determining the number of matched positions in aligned nucleic acid sequences, dividing the number of matched positions by the total number of aligned nucleotides, and multiplying by 100.
  • a matched position refers to a position in which identical nucleotides occur at the same position in aligned nucleic acid sequences.
  • suitable nucleic acid fragments encode polypeptides that are at least about 70% identical, preferably at least about 80% identical to the amino acid sequences described herein.
  • Preferred nucleic acid fragments encode amino acid sequences that are at least about 85% identical to the amino acid sequences described herein.
  • More preferred nucleic acid fragments encode amino acid sequences that are at least about 90% identical to the amino acid sequences described herein.
  • a "homologous protein” is to be understood to comprise proteins which contain an amino acid sequence at least 70% of which, preferably at least 80% of which, most preferably at least 90% of which, corresponds to the amino acid sequence disclosed in (Gish and States, 1993; L37761); wherein corresponds is to be understood to mean that the corresponding amino acids are either identical or are mutually homologous amino acids.
  • the expression "homologous amino acids” denotes those which have corresponding properties, particularly with regard to their charge, hydrophobic character, steric properties, etc.
  • the protein may be from 70% up to less than 100% identical to Acinetobacter sp. ⁇ -ketothiolase (accession no: L37761).
  • Sequence identity of nucleotide or amino acid sequences may be determined conventionally by using known software or computer programs such as the BestFit or Gap pairwise comparison programs (GCG Wisconsin Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. 53711). BestFit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of identity or similarity between two sequences. Gap performs global alignments: all of one sequence with all of another similar sequence using the method of Needleman and Wunsch, J. MoI. Biol. 48:443-453 (1970).
  • the default setting When using a sequence alignment program such as BestFit, to determine the degree of sequence homology, similarity or identity, the default setting may be used, or an appropriate scoring matrix may be selected to optimize identity, similarity or homology scores. Similarly, when using a program such as BestFit to determine sequence identity, similarity or homology between two different amino acid sequences, the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity or homology scores.
  • a sequence alignment program such as BestFit
  • the present invention also relates to plant cells or plants transformed with polynucleotides which contain the complete gene with the polynucleotide sequence corresponding to the phaA gene or fragments thereof, and which can be obtained by screening by means of the hybridization of a corresponding gene bank with a probe which contains the sequence of said polynucleotide molecule or a fragment thereof, and isolation of the DNA sequence.
  • Polynucleotide sequences according to the invention are suitable as hybridization probes for KNA, cDNA and DNA, in order to isolate those cDNAs or genes which exhibit a high degree of similarity to the sequence of the Acinetobacter sp. ⁇ -ketothiolase gene.
  • Polynucleotide sequences according to the invention are also suitable as primers for polymerase chain reaction (PCR) for the production of DNA which encodes an enzyme having aspartate decarboxylase activity.
  • PCR polymerase chain reaction
  • Oligonucleotides such as these, which serve as probes or primers, can contain more than
  • Oligonucleotides with a length of at least 40 or 50 nucleotides are also suitable.
  • isolated means separated from its natural environment.
  • polynucleotide refers in general to polyribonucleotides and polydeoxyribonucleotides, and can denote an unmodified RNA or DNA or a modified RNA or DNA.
  • polypeptides is to be understood to mean peptides or proteins which contain two or more amino acids which are bound via peptide bonds.
  • polypeptides for use in accord with the teachings herein include polypeptides corresponding to Acinetobacter sp. ⁇ -ketothiolase, and also includes those, at least 70% of which, preferably at least 80% of which, are homologous with the polypeptide corresponding to ⁇ -ketothiolase, and most preferably those which exhibit a homology of least 90% to 95% with the polypeptide corresponding to Acinetobacter sp. ⁇ -ketothiolase and which have enzymatic activity.
  • the polypeptides may have a homology of from 70% to up to 100% with respect to Acinetobacter sp. ⁇ - ketothiolase.
  • the invention also relates to transforming plant cells and plants with polynucleotide sequences which result from phaA gene by degeneration of the genetic code.
  • the invention further relates to DNA sequences which hybridize with phaA gene or with parts of phaA gene.
  • conservative amino acid replacements such as the replacement of glycine by alanine or of aspartic acid by glutamic acid in proteins as "sense mutations" which do not result in any fundamental change in the activity of the protein, i.e. which are functionally neutral. It is also known that changes at the N- and/or C-terminus of a protein do not substantially impair the function thereof, and may even stabilize the function.
  • the present invention also relates to employing DNA sequences which hybridize with phaA gene or with parts of phaA gene, or the complements thereof.
  • the present invention relates to DNA sequences which are produced by polymerase chain reaction (PCR) using oligonucleotide primers which result from phaA gene. Oligonucleotides of this type typically have a length of at least 15 nucleotides.
  • stringent conditions or “stringent hybridization conditions” includes reference to conditions under which a polynucleotide will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).
  • stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C, and a wash in 0.1xSSC at 60 to 65° C.
  • the Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with approximately 90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C.
  • Tm thermal melting point
  • moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (Tm).
  • Tm thermal melting point
  • polynucleotide it may be advantageous for propagating the polynucleotide to carry it in a bacterial or fungal strain with the appropriate vector suitable for the cell type.
  • Common methods of propagating polynucleotides and producing proteins in these cell types are known in the art and are described, for example, in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1982) and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989).
  • the polynucleotide sequence introduced into plastids comprises a phaA gene, polynucleotides which are complementary to phaA gene, and polynucleotides which are at least 70%, 80% or 90% identical tophaA gene.
  • Plasmid DNA from Acinetobacter sp coding for the phaA gene was provided by Metabolix (Cambridge, MA). Isolation and amplification of the phaA gene from the native plasmid was performed by polymerase chain reaction (PCR) with the utilization of phaA specific 5' and 3' flanking DNA primers. All primers were designed using the QUICKPRI program of the DNASTAR software.
  • the PCR product was cloned into the vector pCR2.1 S'OTRpsbA, which contained the functional psbA gene promoter and 5' regulatory sequence, by directional cloning after Ndel and Notl restriction digestion of the PCR product and vector.
  • the phaA gene and the 5'UTRpJ 1 Z)A region were sequenced and subsequently cloned into the chloroplast transformation vector pLD-ctv, by directional insertion using appropriate restriction enzymes.
  • the Acinetobacter sp (accession no: L37761, sequence available via NCBI website www.ncbi.nlm.nih.gov) gene, phaA (1179 bp) coding for ⁇ -ketothiolase was amplified by PCR and cloned into the chloroplast transformation vector (pLD-ctv) to finally produce the pLDR-5'UTR-/? ⁇ A vector (Fig IA).
  • the vector contains chloroplast DNA sequences (flanking sequences), which allow site- specific integration by homologous recombination into the inverted repeat region of the chloroplast genome in between the tm ⁇ (tRNA He) and trnA (tRNA Ala) genes (27).
  • aadA aminoglicoside 3'-adenylyltransferase
  • the transcription of the aadA and phaA gene is driven by the constitutive action of the chloroplast 16S ribosomal RNA gene promoter (Prrri), which is located upstream of the aadA gene and should produce dicistrons (aadA-phaA).
  • the psb A gene promoter and 5' regulatory sequence (5' untranslated region; 5' UTR), which is known to enhance translation of foreign genes in the light (28, 29, 30, 31), was used upstream of the phaA gene and should produce phaA monocistron.
  • 5' UTR 5' untranslated region
  • 3'UTR 3' psb A untranslated region
  • Plant Mini Kit (Qiagen, Valencia, CA) was used as the template for PCR reactions.
  • the PCR primer pairs 3P-3M and 4P-4M were used to confirm the integration of the gene cassette into the chloroplast, essentially as described previously (34).
  • Primer pair 5P-2M, 5P-phaA internal and 5P-3'phaA were used to confirm the presence of the phaA gene.
  • PCR analysis was performed using the Gene Amp PCR System 2400 (Perkin Elmer, Chicago).
  • the total plant DNA was obtained from TO and Tl transgenic plants as well as from untransformed tobacco plants using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA) and protocol. Southern blot analyses were performed essentially as described previously (34). Two ⁇ g of plant DNA was restriction digested with Batn ⁇ . and resolved on a 0.8% (w/v) agarose gel at 50 V for 2 h. The gel was soaked in 0.25 N HCl for 15 min and was then rinsed two times with water. The gel was then soaked in transfer buffer (0.4 N NaOH and 1 M NaCl) for 20 min and the denatured DNA was transferred overnight to a nitrocellulose membrane by capillarity.
  • the flanking sequence probe was obtained by BglU/BaniKI restriction digestion of plasmid pUC-ct, which contains the chloroplast flanking sequence (trnl and trnA genes).
  • the phaA probe was obtained by NdeVNotl restriction digestion of plasmid pCR2 ⁇ -5'XJTR-phaA.
  • Probes were radio labeled with 32 P dCTP by using Ready Mix and Quant G-50 micro columns for purification (Amersham, Arlington Heights, EL). Prehybridization and hybridization were performed using the Quick-Hyb solution (Stratagene, La Jolla, Ca). The membrane was washed twice for 15 min at room temperature in 2 X SSC with 0.1% (w/v) SDS, followed by two additional washes at 6O 0 C (to increase the stringency) for 15 min with 0.1 X SSC with 0.1% (w/v) SDS. Radiolabeled blots were exposed to x-ray films and then developed in the Mini-Medical Series x-ray film processor (AFP Imaging, Elmsford, NY).
  • Mini-Medical Series x-ray film processor AFP Imaging, Elmsford, NY.
  • RNA from untransformed and chloroplast transgenic plants was isolated by using the RNeasy Mini Kit (Qiagen, Valencia, CA) and protocol. Northern blot analyses were performed essentially as described previously (49). Total RNA (2.5 ⁇ g) per plant sample was resolved in a 1.2% (w/v) agarose/formaldehyde gel. The phaA probe generation, labeling reaction, prehybridization/hybridization, membrane washing steps, and autoradiography were performed essentially as explained above in the Southern-Blot section.
  • Example 6 Western-Blot Analysis
  • Protein samples were obtained from 100 mg of leaf material from wild type and transgenic lines by grinding the tissue to a fine powder in liquid nitrogen, subsequent homogenization in 200 ⁇ l plant protein extraction buffer (100 mM NaCl, 10 mM EDTA, 200 mM Tris-HCl, 0.05% (w/v) Tween-20, 0.1% (w/v) SDS, 14 mM ⁇ -mercaptoethanol (BME), 400 mM sucrose and 2 mM phenylmethylsulfonyl fluoride) and a centrifugation step at 15.7 x g for 1 minute to remove solids. Protein concentrations were determined by Bradford assay (Bio-Rad Protein Assay) with bovine serum albumin as the protein standard.
  • Proteins were resolved by electrophoresis in a 12% (v/v) SDS-PAGE and then transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was blocked for lhr with PTM buffer: 1 x PBS (phosphate buffer solution), 0.05% (v/v) Tween-20 and 3% (w/v) non-fat dry milk.
  • PTM buffer 1 x PBS (phosphate buffer solution), 0.05% (v/v) Tween-20 and 3% (w/v) non-fat dry milk.
  • the membrane was probed for 2 hrs with rabbit anti- ⁇ -ketothiolase antibody (Metabolix, Cambridge, MA) in a dilution of 1:1,000, then rinsed with water twice and probed with alkaline phosphatase-conjugated secondary antibody (goat anti-rabbit, Sigma) for 1.5 hrs in a 1:20,000 dilution. Finally, the membrane was washed 3 times for 10 minutes with PT buffer (Ix PBS, 0.05% (v/v) Tween- 20) and one time with 1 x PBS, followed by incubation in Lumi-phos WB (Pierce, Rockford, IL) reagent for the alkaline phosphatase reaction. Film exposure took place for 3 minutes.
  • PT buffer Ix PBS, 0.05% (v/v) Tween- 20
  • Lumi-phos WB Pierford, IL
  • Protein samples were obtained by grinding 1 g of leaf tissue to a fine powder in liquid nitrogen, followed by the addition of 2 ml ice cold ⁇ -ketothiolase extraction buffer (100 mM Tris-HCl pH 8.1, 50 mM MgCl 2 , 5 mM BME) and homogenization. The homogenates were centrifuged for 10 minutes at 4°C at 5,000 g, and the supernatant was passed through PD-10 columns (Amersham, Arlington Heights, IL) containing Sephadex G-25 M for desalting, and elution was optimized for the recovery of proteins of size range 25 to 60 kDa.
  • 2 ml ice cold ⁇ -ketothiolase extraction buffer 100 mM Tris-HCl pH 8.1, 50 mM MgCl 2 , 5 mM BME
  • the homogenates were centrifuged for 10 minutes at 4°C at 5,000 g, and the supernatant was passed through PD-10 columns (Amersham
  • Protein concentration was determined by a Bradford assay, ⁇ - ketothiolase activity was measured spectrophotometrically at 304 nm in the thiolysis direction (breaking down acetoacetyl-CoA to acetyl-CoA) by monitoring the disappearance of acetoacetyl-CoA for 60 seconds, which in the presence of Mg ion forms a magnesium enolate with absorbance at 304 nm; this protocol is an adaptation of the protocol by Senior and Davis (55).
  • the reaction took place in a total volume of 1 ml containing 62.4 mM Tris-HCl pH 8.1, 50 mM MgCl 2 , 62.5 ⁇ M CoA, 62.5 ⁇ M Acetoacetyl-CoA (substrate is dissolved in 50 mM phosphate buffer pH 4.7), 10 ⁇ l of plant extract ( ⁇ - ketothiolase sample), and bringing the volume with distilled water to 1 ml.
  • the plant extract containing the ⁇ -ketothiolase was added at the end immediately before the sample reading.
  • the enzyme specific activity is given in units per mg of total plant protein and 1 unit is defined as the degradation of 1 ⁇ mol/min of acetoacetyl-CoA under standard reaction conditions.
  • % (v/v) glutaraldehyde in phosphate buffer for 12 hours at room temperature applying a continuous vacuum for the first 3 hrs of incubation and degassing (by bringing the vacuum up and down slowly) for 10 min at 1 hr, 2hr and 3 hr.
  • the fixed anthers were dehydrated in an ethanol series (5%, 10 % to 80 % in increments of 10, 95 % and 100 %) for 30 min per gradient treatment. Samples were kept overnight in fresh 100 % ethanol and were washed twice the next day for 1 hr in 100 % ethanol.
  • Samples were then treated with a gradient (25 % to 100%) of Citro SoIv clearing (Fisher, Pittsburgh, PA) reagent for 30 min per gradient treatment, and finally embedded in Paraplast Plus (Fisher, Pittsburgh, PA).
  • Tissue embedding was performed in molten paraffin for 3 days, changing the molten paraffin every 8 to 12 hrs. Paraffin treated tissue was finally embedded into paraffin blocks by using the Leica EG 1160 paraffin embedding station (Leica, Solms).
  • Leica, Solms Leica, Solms
  • a metal blade microtome, model HM 315 (MICROM, Walldorf) was used for tissue embedded sectioning.
  • tissue sections were put onto Superfrost/Plus microscope slides, followed by a rehydration step and tissue staining with 0.05 % (w/v) toluidine blue.
  • Tissue slides were observed under the Olympus BX60F5 light microscope and Olymppus U-CMAD-2 camera (Olympus, Melville, NY). Flower developmental stages were characterized following the procedure described by Koltunow et al (56).
  • transgenic plants were moved to a separate growth chamber after the first indication of flower bud formation and were kept away from any contact with wild type and other transgenic lines; the flowers were covered with thin transparent plastic bags to inhibit any possibility of cross pollination. Bags were only removed to take pictures.
  • Transgenic plants were kept under continuous illumination for 10 days with a photon flux density of 11,250 ⁇ Em-2 supplied throughout this period. The number of flowers developed was counted daily throughout these ten days, while newly formed flowers, senescent flowers, and fallen flowers were recorded. The development of fruit capsules and seeds were also counted. After the 10 days, a 16 hrs light/ 8 hrs dark photoperiod was reestablished, while the plants were kept from contact with any other plant for 20 days to allow maturation of the fruit capsules and to harvest seeds produced during continuous illumination.
  • Chloroplast transgenic plants were obtained through particle bombardment following the method described previously (33, 34, 35). More than 10 positive independent transgenic lines were obtained. Several independent transgenic lines were characterized, confirming that independent chloroplast transgenic lines show little variation in foreign gene expression (26). PCR based analysis with the primer pairs, 3P and 3M and 4P-4M were used to test the integration of the transgene construct into the chloroplast genome (36). The 3P and 4P primers land on the native chloroplast genome, upstream of the gene cassette, and the 3M and 4M primers land on the aadA gene, which is located within the gene cassette (Fig IA).
  • a PCR product of 1.65 kb should be obtained; this product was detected in transgenic lines (Fig 2A lanes 2, 3). Untransformed plants as well as mutant plants, which had undergone spontaneous mutation of the 16S rRNA gene and acquired resistance to spectinomycin, did not show any PCR product, indicating that these plants are negative for integration (Fig 2A lane 1).
  • the integration of the aadA gene, as well as the phaA was further confirmed by the use of primer pairs 5P-2M, 5P-3'phaA, and 5P-phaAinternal, which produce PCR products of sizes 3.56 kb, 2.0 kb and 1.5 kb, respectively. These primers anneal at different locations within the gene construct (see Fig IA). Results revealed specific PCR size products in the transgenic lines (Fig 2A lanes 4-6), confirming the presence of the pha A gene.
  • DNA from untransformed plants and transgenic lines were digested with BarriHI (Fig IA and B) and probed with either a flanking sequence probe or a pha A probe, resulting in the detection of a 10 kb transgenic chloroplast fragment (Fig 2B-E, lanes 2-4).
  • the monocistron was not detected with the aadA probe, showing that polycistrons are transcribed from the engineered Prrn promoter; less abundant polycistrons transcribed from the native 16S promoter were also detected (Fig. 2G).
  • Fig. 2G shows that both the monocistron and dicistron transcripts were abundant in the transgenic plants, because of the efficiency of the psbA and Vrrn promoters, which are strong endogenous promoters.
  • larger size transcripts were detected, one of them correlating with the size of a transcript starting at the chloroplast native 16S promoter (Prrn) and tenninating at the 3' UTR psbA; the predicted size of this transcript is 4,723 nt.
  • Other transcripts detected may be either read through (because 3' UTR does not terminate transcription efficiently in chloroplast) or processed products.
  • the activity of the chloroplast-expressed ⁇ -ketothiolase was measured in the thiolysis direction (breaking down acetoacetyl-CoA to acetyl-CoA) spectrophotometrically at 304 nm.
  • the chloroplast transgenic lines showed ⁇ -ketothiolase activities that were up to 30-fold higher than previous levels demonstrated from nuclear transgenic plants. No endogenous ⁇ -ketothiolase activity was detected in untransformed tobacco plants (less than 0.0001 unit/mg plant protein; Table 1).
  • the chloroplast transgenic lines showed levels of activity that reached 14.08 to 14.71 during normal photoperiod (16hligh/8hr dark, Table I).
  • Enzyme activity for transgenic lines 4A and 4B and untransformed tobacco plants at the respective illumination periods are shown, Protein samples were obtained by grinding 1 g of leaf tissue in liquid nitrogen, followed by the addition of /3-ketotbiolase extraction buffer (100 HIM Tris-HGi, pH 8,1, 50 ⁇ TM MgCU, 5 HIM BME) and homogenizat ⁇ on. Total plant protein concentration was determined by Bradford assay. Ten microliters of crude plant extract was used per assay. J3-Ketothiolase activity was measured spectrophotometry cally at 304 nm for 60 s in the thiolysls direction, na, Hot applicable.
  • Chlorophyll content analysis of three independent T 1 transgenic lines showed average chlorophyll content of 1.90 ⁇ 0.12 mg/g fresh weight.
  • the chlorophyll content of three wild type plants average, 1.92 ⁇ 0.20 mg/g fresh weight.
  • the chloroplast transgenic lines showed specific defects in anther development and failed to produce viable pollen.
  • the anthers were characterized by the lack of pollen grains (Fig 4H) or when pollen grains were formed, they were abnormal with a collapsed phenotype (Fig 6). Additionally, the stamens were shorter (Fig 4F) than in wild type plants (Fig 4G), adding a degree of severity to the male sterile phenotype.
  • Fig 4H the stamens were shorter than in wild type plants
  • Fig 4G wild type plants
  • a total of 15 emasculated wild type flowers from three untransformed plants were pollinated with pollen collected from anthers from transgenic lines 4A, 4B, and 4C.
  • Plastids in anthers may be in low abundance when compared to the numbers in leaves, but they produce enough ⁇ -ketothiolase to affect pollen development in anthers.
  • Fig. 7A flowers and specifically anthers were green during anther development (stage 1 of flower development is shown). This means that chloroplasts are present and are metabolically active.
  • Northern-blot analysis of leaves showed that the mRNAs coding for ⁇ -ketothiolase are found in monocistronic and polycistronic form, the monocistron form being the most abundant transcript (Fig 2F). The same pattern was maintained in the phaA transcripts in flowers and anthers (Fig 7B).
  • ⁇ -ketothiolase was detected by western-blot in both whole flowers and anthers from transgenic plants, confirming that the ⁇ -ketothiolase was present during anther development, and should play a role in the male sterility phenotype (Fig 7C).
  • transgenic anthers showed abnormal development of the epidermis and endothecium, probably resulting in the aberrant shape of anthers.
  • the anthers of the transgenic lines at stage —2 were also advanced in the aberrant phenotype with the microspores already separated, a developmental step that should have been observed at stage +2. Again the tapetal layer was broken.
  • stage -5 which is a very early stage of flower development, showed great similarities with stage -2 in the wild type plants, but aberrant pattern of tissue development could still be observed.
  • stage -1 in transgenic line the tapetum was shrunken and discontinuous and formation of pollen grains was evident at stage 1.
  • stage -1 and +1 morphological changes observed in stage -1 and +1 should be observed at much later stages, +3 and +4.
  • stages -5 to +1 transgenic lines showed accelerated anther development, which averaged +3 stages ahead from the wild type plants.
  • stage accelerated phenotype increased even more, at an average of 4 to 6 stages ahead of wild type.
  • stage +2 in the transgenic lines cells adjacent to the stomium had degenerated and only remnants of the tapetum were observed.
  • the thickening of the outer wall is accompanied by enlarged endothecium and vacuolation, which greatly decreased the inner space of the locules, crushing pollen grains and resulted in the irregular shape and collapsed phenotype.
  • Anther development is a very complex process involving the coordination of several genes and the specific development and maturation of several tissues and cells (13); any defect in these well-coordinated processes may lead to dysfunctional pollen.
  • Many male-sterility systems produced by mutations or nuclear expressions of foreign proteins have shown to interfere with the function or differentiation of tapetum, indicating that this tissue is essential for the production of viable pollen (17).
  • the tapetum of the transgenic lines was severely impaired. The tapetum is critical

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Abstract

L'invention concerne des procédés permettant de parvenir à une stérilité mâle de plantes. L'invention illustre tout particulièrement la transformation du génome plastidial avec un vecteur exprimant le gène phaA. L'expression du gène phaA dans des plastes donne lieu à des plantes ne manifestant pas d'effets pléiotropiques, à l'exception de la stérilité mâle. L'invention concerne également des plantes et des cellules transformées stables, de même que des exemples de vecteurs pour exprimer le gène phaA dans des plastes.
PCT/US2005/032402 2004-09-01 2005-09-13 Genie genetique utilise pour parvenir a la sterilite male de plantes WO2006031721A2 (fr)

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CN111549057A (zh) * 2020-05-28 2020-08-18 潍坊兴旺生物种业有限公司 一种利用番茄雄性不育基因及可见连锁标记创制雄性不育系的方法

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DE60040125D1 (de) * 1999-08-09 2008-10-16 Riken Wako Verfahren zur Transformierung von Pflanzen, transformierte Pflanzen und Verfahren zur Herstellung von Polyestern

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CN111549057A (zh) * 2020-05-28 2020-08-18 潍坊兴旺生物种业有限公司 一种利用番茄雄性不育基因及可见连锁标记创制雄性不育系的方法
CN111549057B (zh) * 2020-05-28 2021-11-30 潍坊兴旺生物种业有限公司 一种利用番茄雄性不育基因及可见连锁标记创制雄性不育系的方法

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