RESUMO
We used two genes, TMV-CP and PPI1 (pepper-PMMV interaction 1 transcription factor), to transform commercially important chili pepper (Capsicum annuum) inbred lines (P915, P409) by means of Agrobacterium co-culture. Eighteen independently transformed T0 plants were obtained. The most critical point in the pepper transformation protocol was the selection of shoots growing on calli--referred to as callus-mediated shoot formation (indirect shooting)--because shoots not grown from the callus (direct shooting from the wounded surface) developed into non-transformants. Selection of the correct right callus type also proved to be an important requirement for obtaining transformed peppers. Six different types of callus developed during the selection process. Shoots regenerated from two of these types, while one type regenerated significantly more shoots than the other types, suggesting that the capacity for shoot formation is callus type-specific. Although the transformation rate was low, transformation via callus-mediated shoot formation proved to be reproducible and was confirmed by Southern and Northern blot analyses. Based on the experimental data, we have succeeded in developing a new protocol for the selection and transformation of pepper and expect that it will be used in the future for pepper transformation.
Assuntos
Capsicum/genética , Engenharia Genética/métodos , Vetores Genéticos/genética , Plantas Geneticamente Modificadas/genética , Rhizobium/genética , Transformação Genética/genética , Capsicum/crescimento & desenvolvimento , Capsicum/microbiologia , Células Cultivadas , Técnicas de Cocultura/métodos , Imunidade Inata/genética , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/microbiologia , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/microbiologia , Vírus do Mosaico do Tabaco/genéticaRESUMO
Three cDNAs (iAGPLI-1, iAGPLI-2, and iAGPLI-3) encoding an isoform of AGPase large subunit were isolated from a sweet potato cDNA library constructed from tuberous root tissue. iAGPLI-1 was 2,161 bp in length and contained an open reading frame of 517 amino acids with a calculated molecular mass of 57,689 Da. iAGPLI-2 and iAGPLI-3 were 1,804 and 1,524 bp in length, respectively, and contained partial open reading frames of 490 and 385 amino acids. Deduced amino acid sequence comparison analysis showed that iAGPLI-1 has sequence identity with iAGPLI-2 (97.9) and iAGPLI-3 (98.7%) while iAGPLI-2 and iAGPLI-3 have 96.8% sequence identity. iAGPLI-1 had the highest sequence identity of 77.8% with potato AGPase (sAGPL1). Steady-state levels of iAGPLI-1 transcripts were expressed predominantly in the stem, and moderately in the tuberous root, but not in either the roots or leaves. However, AGPase activity was present in all tissues. The expression level in the stem declined dramatically after a 12 h incubation in the dark to nearly 3% of the value under light, although the activity under a dark condition remained at half the levels in light. The activity levels were not correlated with the transcript levels. iAGPL transcripts in leaves were induced by sucrose feeding but not by glucose or fructose. Therefore, the expression of iAGPLI-1 is regulated in stem tissue preferentially and by sucrose. Southern blot analysis showed that the sweet potato genome contained several copies of iAGPLI gene probably due to polyploidy.
Assuntos
DNA Complementar/genética , Nucleotidiltransferases/genética , Solanaceae/genética , Sequência de Aminoácidos , Northern Blotting , Southern Blotting , DNA Complementar/química , DNA de Plantas/genética , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glucose-1-Fosfato Adenililtransferase , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Nucleotidiltransferases/metabolismo , Subunidades Proteicas , RNA de Plantas/genética , RNA de Plantas/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Solanaceae/enzimologia , Distribuição TecidualRESUMO
cDNA clones encoding sweet potato AGPase large subunit (iAGPLI) from the cDNA library constructed from the tuberous root were isolated. Two clones were characterized and named iAGPLI-a and iAGPLI-b. They were 1,661 bp and 1,277 bp in length and contained partial open reading frames of 450 and 306 amino acids, respectively. Both nucleic acid and amino acid sequence identities between iAGPLI-a and iAGPLI-b were 83.8% and 97.3%, respectively. Based on the amino acid sequence analysis, iAGPLI-a and iAGPLI-b share the highest sequence identity (81%) with potato AGPase large subunit. The iAGPLI-a and iAGPLI-b genes were expressed predominantly in the stem and weakly in the tuberous root, and no transcript was expressed in other tissues. The sweet potato genome contains several copies of the iAGPLI gene.
Assuntos
DNA Complementar/isolamento & purificação , Nucleotidiltransferases/genética , Solanaceae/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , DNA Complementar/química , DNA Complementar/genética , DNA de Plantas/genética , Dosagem de Genes , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glucose-1-Fosfato Adenililtransferase , Isoenzimas/genética , Dados de Sequência Molecular , Filogenia , Polimorfismo Genético , RNA de Plantas/genética , RNA de Plantas/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Solanaceae/enzimologia , Distribuição TecidualRESUMO
The plastid ribosomal protein s16 (rps16) gene was cloned from potato (Solanum tuberosum L. ssp. tuberosum cv Desiree) by PCR amplification to obtain a new homologous recombination site of plastid transformation. The potato rps16 genomic clone was 1627 bp in size and the coding region was interrupted by an 859 bp intron. Exon I was 40 bp, encoding 13 amino acids and exon II was 227 bp, encoding a 76 amino acid polypeptide. The nucleotide sequence of the rps16 gene from the "Désirée" potato shared perfect identity with the sequence from the "Superior" potato in the coding region. Three nucleotide substitutions, two nucleotide insertions, and one nucleotide deletion were found between the intron sequence of both "Désirée" and "Superior" cultivars. The amino acid sequence of the potato rps16 gene showed a high level of identity with rice, maize, tobacco, and mustard (84-94%) and a relatively low level compared with Bacillus stearothermophilus and E. coli (27-28%). Expression of the rps16 gene was strong in chloroplasts and transcripts were detectable in amyloplasts, suggesting that the rps16 gene is active in nonphotosynthetic plastids as well as in photosynthetic plastids. These results indicate that the potato rps16 gene can be used as a new homologous recombination site of plastid transformation for potato cultivars.