RESUMO
The maize P gene, which specifies red pigmentation of the kernel pericarp, cob, and other floral organs, has been an important model since the early days of modern genetics. Here we show that P encodes a Myb homolog that recognizes the sequence CCT/AACC, in sharp contrast with the C/TAACGG bound by vertebrate Myb proteins. P binds to and activates transcription of the A1 gene required for 3-deoxy flavonoid and phlobaphene biosynthesis, but not the Bz1 gene required for anthocyanin biosynthesis. The maize C1 gene, which also encodes a Myb homolog, activates both the A1 and Bz1 genes, but only in the presence of a basic-helix-loop-helix coactivator encoded by the maize genes R or B. These results indicate that Myb homologs can differentially regulate gene expression by binding different DNA sequences, through combinatorial interactions with other factors, or both.
Assuntos
Proteínas de Ligação a DNA/genética , Flavonoides/biossíntese , Genes de Plantas , Genes Reguladores , Oncogenes , Proteínas de Plantas/genética , Zea mays/genética , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Dados de Sequência Molecular , Mutagênese , Proteínas Oncogênicas v-myb , Pigmentos Biológicos/biossíntese , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Proteínas Oncogênicas de Retroviridae/biossíntese , Proteínas Oncogênicas de Retroviridae/genética , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , Vertebrados , Zea mays/fisiologiaRESUMO
The pl gene encodes a regulatory protein that controls the transcription of a number of structural genes of the anthocyanin biosynthetic pathway in maize. pl alleles have been classified phenotypically into two categories: dominant (Pl) alleles lead to intense, light-independent pigmentation in vegetative and floral organs of the plant; recessive "sun-red" alleles (pl) lead to light-dependent red pigmentation in which only tissues exposed to light become pigmented. Based on these observations, two alternate pathways leading to anthocyanin synthesis in the plant have been proposed: one requiring light and the other bypassing the light requirement through the action of Pl. To evaluate this hypothesis, we have analyzed light-independent and light-dependent alleles of pl. Sequence analysis revealed that the two types of alleles have very distinct promoters but have the capacity to encode very similar proteins. The protein encoded by one recessive allele was shown to be functional in transient assays. Measurements of husk mRNA levels by quantitative polymerase chain reaction showed that sun-red pl alleles are expressed at much lower levels than a Pl allele, but their expression is increased approximately sixfold by exposure to light. These results lead to the conclusion that the sun-red pl alleles are not null; instead, they synthesize functional mRNA and protein. We propose that the light-dependent pigmentation observed in pl plants is the result of a threshold effect in which light exposure boosts pl mRNA expression past a crucial level necessary to generate sufficient PL protein molecules to activate transcription of the anthocyanin structural genes.
Assuntos
Antocianinas/genética , Genes de Plantas , Zea mays/genética , Alelos , Sequência de Aminoácidos , Antocianinas/biossíntese , Sequência de Bases , Clonagem Molecular , DNA/genética , Expressão Gênica/efeitos da radiação , Genes de Plantas/efeitos da radiação , Genes Reguladores/efeitos da radiação , Luz , Dados de Sequência Molecular , Pigmentação/genética , Pigmentação/efeitos da radiação , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Zea mays/metabolismo , Zea mays/efeitos da radiaçãoRESUMO
Transgenic tobacco plants and progeny carrying coding sequences for neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) were recovered following microprojectile bombardment of tobacco leaves. Transgenic plants were regenerated from bombarded leaf pieces of tobacco cvs. 'Xanthi' and 'Ky 17' which were cultured in the presence of 100 or 200 micrograms/ml kanamycin for six to eight weeks. Among 160 putative transgenic plants from at least 16 independent transformation events 76% expressed NPTII, and 50% expressed GUS. Southern analysis of plants expressing either one or both of the enzymes indicated DNA in high molecular weight DNA in 8 of 9 independent transformants analyzed. Two independent transformants and their progeny were analyzed in detail. Analysis of progeny for quantitative enzyme levels of NPTII and GUS, and Southern analysis of parents and progeny clearly demonstrated that the genes were transmitted to progeny. One transformant demonstrated Mendelian ratios for seed germination on kanamycin-containing medium while the other transformant had non-Mendelian ratios. DNA analysis of progeny indicate complex integration of the plasmid DNA, and suggest that rearrangements of this DNA has occurred. These results are consistent with other methods of direct DNA uptake into cells, and verify that the microprojectile bombardment method is capable of DNA delivery into intact plant cells which can give rise to transgenic plants and progeny.
Assuntos
Glucuronidase/genética , Nicotiana/genética , Fosfotransferases/genética , Plantas Tóxicas , Transformação Genética , Southern Blotting , DNA/genética , DNA/isolamento & purificação , Engenharia Genética/métodos , Vetores Genéticos , Glucuronidase/metabolismo , Canamicina/farmacologia , Canamicina Quinase , Fosfotransferases/metabolismo , PlasmídeosRESUMO
Protoplasts of a Schizophyllum commune tryptophan auxotroph (trp1), deficient in indole-3-glycerol phosphate synthetase (IGPS), were transformed to trp+ with plasmid DNA containing the Schizophyllum TRP1 sequence. Efficiencies up to 30 transformants per microgram of plasmid DNA were obtained. Southern blots reveal that the transforming DNA is integrated in chromosomal DNA. The trp+ phenotype of transformants is stable in meiosis and mitosis. Transformants possess IGPS activity comparable to wild-type cells.
