RESUMEN
Plastids and mitochondria, the DNA-containing cytoplasmic organelles, are maternally inherited in the majority of angiosperm species. Even in plants with strict maternal inheritance, exceptional paternal transmission of plastids has been observed. Our objective was to detect rare leakage of plastids via pollen in Nicotiana sylvestris and to determine if pollen transmission of plastids results in co-transmission of paternal mitochondria. As father plants, we used N. sylvestris plants with transgenic, selectable plastids and wild-type mitochondria. As mother plants, we used N. sylvestris plants with Nicotiana undulata cytoplasm, including the CMS-92 mitochondria that cause cytoplasmic male sterility (CMS) by homeotic transformation of the stamens. We report here exceptional paternal plastid DNA in approximately 0.002% of N. sylvestris seedlings. However, we did not detect paternal mitochondrial DNA in any of the six plastid-transmission lines, suggesting independent transmission of the cytoplasmic organelles via pollen. When we used fertile N. sylvestris as mothers, we obtained eight fertile plastid transmission lines, which did not transmit their plastids via pollen at higher frequencies than their fathers. We discuss the implications for transgene containment and plant evolutionary histories inferred from cytoplasmic phylogenies.
Asunto(s)
ADN Mitocondrial/genética , Herencia Extracromosómica/genética , Mitocondrias/genética , Nicotiana/genética , Plastidios/genética , Polen/genética , Citoplasma/genética , ADN de Plantas/genética , Genes Mitocondriales/genética , Marcadores Genéticos , Genotipo , Brotes de la Planta , Plantones/genética , TransgenesRESUMEN
Plastids in Nicotiana tabacum are normally transmitted to the progeny by the maternal parent only. However, low-frequency paternal plastid transmission has been reported in crosses involving parents with an alien cytoplasm. Our objective was to determine whether paternal plastids are transmitted in crosses between parents with the normal cytoplasm. The transplastomic father lines carried a spectinomycin resistance (aadA) transgene incorporated in the plastid genome. The mother lines in the crosses were either (i) alloplasmic, with the Nicotiana undulata cytoplasm that confers cytoplasmic male sterility (CMS92) or (ii) normal, with the fertile N. tabacum cytoplasm. Here we report that plastids from the transplastomic father were transmitted in both cases at low (10(-4)-10(-5)) frequencies; therefore, rare paternal pollen transmission is not simply due to breakdown of normal controls caused by the alien cytoplasm. Furthermore, we have found that the entire plastid genome was transmitted by pollen rather than small plastid genome (ptDNA) fragments. Interestingly, the plants, which inherited paternal plastids, also carried paternal mitochondrial DNA, indicating cotransmission of plastids and mitochondria in the same pollen. The detection of rare paternal plastid transmission described here was facilitated by direct selection for the transplastomic spectinomycin resistance marker in tissue culture; therefore, recovery of rare paternal plastids in the germline is less likely to occur under field conditions.
Asunto(s)
Contención de Riesgos Biológicos , Patrón de Herencia/genética , Mitocondrias/genética , Nicotiana/genética , Plastidios/genética , Polen/genética , Transgenes , Cruzamientos Genéticos , Citoplasma/genética , Sondas de ADN , ADN de Cloroplastos/genética , ADN Mitocondrial/genética , Resistencia a Medicamentos , Infertilidad Vegetal , Polimorfismo de Longitud del Fragmento de Restricción , Selección GenéticaRESUMEN
We report here the control of potato tuber moth (Phthorimaea operculella) by incorporating a truncated Bacillus thuringiensis cry9Aa2 gene in the plastid genome. Plasmids pSKC84 and pSKC85 are derivatives of a new polycistronic plastid transformation vector, pPRV312L, that carries spectinomycin resistance (aadA) as a selective marker and targets insertions in the trnI-trnA intergenic region. The Cry9Aa2 N-terminal region (82.1 kDa; 734 amino acids) was expressed in a cassette, which consists of 49 nucleotides of the cry9Aa2 leader and the 3'-untranslated region of the plastid rbcL gene (TrbcL), and relies on readthrough transcription from the plastid rRNA operon. In a tobacco leaf bioassay, expression of Cry9Aa2 conferred resistance to potato tuber moth. In accordance, the Cry9Aa2 insecticidal protein accumulated to high levels, approximately 10% of the total soluble cellular protein and approximately 20% in the membrane fraction. However, high-level Cry9Aa2 expression significantly delayed plant development. Thus, a practical system to control potato tuber moth by Cry9Aa2 expression calls for down-regulation of its expression.
Asunto(s)
Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Cloroplastos/metabolismo , Endotoxinas/genética , Proteínas Hemolisinas/genética , Mariposas Nocturnas/metabolismo , Nicotiana/genética , Control Biológico de Vectores/métodos , Enfermedades de las Plantas/genética , Plantas Modificadas Genéticamente , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Bioensayo , Endotoxinas/metabolismo , Técnicas Genéticas , Vectores Genéticos , Proteínas Hemolisinas/metabolismo , Insectos , Plastidios/metabolismo , Solanum tuberosum , Nicotiana/metabolismoRESUMEN
Fragment C (TetC) is a non-toxic 47 kDa polypeptide fragment of tetanus toxin that can be used as a subunit vaccine against tetanus. Expression of TetC in Escherichia coli and yeast was dependent on the availability of synthetic genes that were required to improve translation efficiency and stabilize the mRNA. To explore the feasibility of producing TetC in tobacco leaves, we attempted expression of both the bacterial high-AT (72.3% AT) and the synthetic higher-GC genes (52.5% AT) in tobacco chloroplasts. We report here that the bacterial high-AT mRNA is stable in tobacco chloroplasts. Significant TetC accumulation was obtained from both genes, 25 and 10% of total soluble cellular protein, respectively, proving the versatility of plastids for expression of unmodified high-AT and high-GC genes. Mucosal immunization of mice with the plastid- produced TetC induced protective levels of TetC antibodies. Thus, expression of TetC in chloroplasts provides a potential route towards the development of a safe, plant-based tetanus vaccine for nasal and oral applications.