RESUMO
The genomic and cDNA sequences of BnSUT1C were isolated from B. napus. Combination of cDNA and genomic DNA sequences revealed that the BnSUT1C gene contained three exons and two introns. The cDNA encodes a protein of 513 amino acids with a calculated molecular mass of 54.7 kDa and an isoelectric point of 9.12. It exhibits typical features of sucrose transporter with 12 trans-membranes spanning domains. BnSUT1C showed highly homologous with AtSUC1 and AtSUC5. A histidine residue, which is conserved across all functional sucrose transporter proteins in higher plants, is located at position 66 of the BnSUT1C. Two putative pollen-specific cis-elements, AGAAA and GTGA motifs, are located in 5′-upstream of BnSUT1C. The spatial and temporal expression patterns carried out by semi-quantitative RT-PCR and Real-Time PCR, which indicated that BnSUT1C predominantly expressed in later developmental stages of anther, as tapetal cells began to shrink and collapse. BnSUT1C could mediate the uptake of sucrose in the pollen and retrieval of tapetal degenerated products during pollen maturation.
Assuntos
Sequência de Aminoácidos , Arabidopsis/genética , Sequência de Bases , Brassica napus/genética , Clonagem Molecular , DNA/genética , DNA Complementar/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/isolamento & purificação , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , RNA Mensageiro/genética , Homologia de Sequência de Aminoácidos , TranscriptomaRESUMO
Background: For successful in vitro plant regeneration, plant cell lines with multiple transgene integration and low transgene expression levels need to be ruled out. Although real-time polymerase chain reaction (RT-PCR) is a rapid way to accomplish this, it is also expensive and typically limits the size of the target sequence. Quantitative competitive PCR (QC-PCR) is proven to be a safe and accurate method for determination of both copy number and quantification of transcript levels of synthetic transgenes in transformed plants. Results: The glyphosate oxidoreductase genewas chemically synthesized and used to transform Brassica napus L. via Agrobactrium-mediated transformation. A construct containing the mutated form of a synthetic glyphosate oxidoreductase (gox) gene (internal standard) was prepared. Gene copy number was estimated in nine independent transgenic lines using QC-PCR as well as the standard method of Southern blot analysis. By quantitative RT-PCR, transcript levels were also determined in these lines. High (> 3), medium to high (2.2-3), medium to low (1-2.2), and low (< 1) levels of transcript were detected. Conclusions: No direct relationship was found between copy number and transgene expression levels. QC-PCR method could be implemented to screen putative transgenic plants and quickly select single T-DNA inserts. QC-PCR methods and the prepared competitor construct may be useful for future quantification of commercial transgenic food and feed.
Assuntos
Brassica napus/enzimologia , Brassica napus/genética , Glicina/genética , Oxirredutases/genética , Reação em Cadeia da Polimerase/métodos , Glicina/análogos & derivados , Glicina/fisiologia , Oxirredutases/fisiologia , Transcrição Gênica , TransgenesRESUMO
Analysis of genetic main effects and genotype x environment (GE) interaction effects on the oil content of oilseed rape (Brassica napus L.) was conducted by using a genetic model for the quantitative traits of seeds in diploid plants. The experiments were carried out over two years with 8 parents and a diallel mating design, which produced F1 and F2 generations. We found that the oil content of rape was simultaneously controlled by embryo genetic effect, cytoplasmic effects and maternal genetic effect as well as GE interaction effects, with the cytoplasmic and maternal effects playing the main role. The results indicate that selection of maternal plants for high oil content would be more efficient than selection based on single seeds. Since the GE interaction effects accounted for 73.69 percent of the total genetic effects and were more important than the genetic main effects it seems that selection might be influenced by environmental conditions. The estimate of narrow-sense heritability for oil content was 73.52 percent with the interaction heritability being larger than the general heritability, indicating that the early generations can be used for selection for high oil content. The prediction of genetic effects suggested that the parent cultivars Youcai 601 and Gaoyou 605 were better than the other cultivars for increasing oil content during the breeding of B. napus. The implications for the quantitative trait loci mapping of seed traits interfered by these three genetic systems is also discussed.