RESUMO
BACKGROUND: Chlamydomonas reinhardtii is an unicellular green alga used for functional genomics studies and heterologous protein expression. A major hindrance in these studies is the low level and instability of expression of nuclear transgenes, due to their rearrangement and/or silencing over time. RESULTS: We constructed dedicated vectors for Agrobacterium-mediated transformation carrying, within the T-DNA borders, the Paromomycin (Paro) selectable marker and an expression cassette containing the Luciferase (Luc) reporter gene. These vectors and newly developed co-cultivation methods were used to compare the efficiency, stability and insertion sites of Agrobacterium- versus electroporation-mediated transformation. The influence of different transformation methods, of the cell wall, of the virulence of different Agrobacterium strains, and of transgene orientation with respect to T-DNA borders were assessed. False positive transformants were more frequent in Agrobacterium-mediated transformation compared to electroporation, compensating for the slightly lower proportion of silenced transformants observed in Agrobacterium-mediated transformation than in electroporation. The proportion of silenced transformants remained stable after 20 cycles of subculture in selective medium. Next generation sequencing confirmed the nuclear insertion points, which occurred in exons or untraslated regions (UTRs) for 10 out of 10 Agrobacterium-mediated and 9 out of 13 of electroporation-mediated insertions. Electroporation also resulted in higher numbers of insertions at multiple loci. CONCLUSIONS: Due to its labor-intensive nature, Agrobacterium transformation of Chlamydomonas does not present significant advantages over electroporation, with the possible exception of its use in insertional mutagenesis, due to the higher proportion of within-gene, single-locus insertions. Our data indirectly support the hypothesis that rearrangement of transforming DNA occurs in the Chlamydomonas cell, rather than in the extracellular space as previously proposed.
Assuntos
Agrobacterium/genética , Chlamydomonas reinhardtii/genética , Eletroporação/métodos , Transformação Genética , DNA Bacteriano , Regulação da Expressão Gênica de Plantas , Genes Reporter , Marcadores Genéticos , Vetores Genéticos , Genoma de Planta , Luciferases de Renilla/genética , Plantas Geneticamente Modificadas , TransgenesRESUMO
Ripe tomato fruits accumulate significant amounts of the linear carotene lycopene, but only trace amounts of xanthophylls (oxygenated carotenoids). We overexpressed the lycopene beta-cyclase (b-Lcy) and beta-carotene hydroxylase (b-Chy) genes under the control of the fruit-specific Pds promoter. Transgene and protein expression was followed through semi-quantitative reverse transcription-PCR, Western blotting, and enzyme assays. Fruits of the transformants showed a significant increase of beta-carotene, beta-cryptoxanthin and zeaxanthin. The carotenoid composition of leaves remained unaltered. The transgenes and the phenotype are inherited in a dominant Mendelian fashion. This is the first example of successful metabolic engineering of xanthophyll content in tomato fruits.
Assuntos
Engenharia Genética/métodos , Liases Intramoleculares/genética , Oxigenases de Função Mista/genética , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Xantofilas/metabolismo , beta Caroteno/análogos & derivados , Arabidopsis/genética , Western Blotting , Criptoxantinas , Frutas/química , Frutas/metabolismo , Expressão Gênica , Genes Dominantes , Liases Intramoleculares/metabolismo , Solanum lycopersicum/química , Oxigenases de Função Mista/metabolismo , Fenótipo , Folhas de Planta/química , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transgenes , Xantofilas/análise , Zeaxantinas , beta Caroteno/análise , beta Caroteno/metabolismoRESUMO
Cryptochromes are blue light photoreceptors found in plants, bacteria, and animals. In Arabidopsis, cryptochrome 2 (cry2) is involved primarily in the control of flowering time and in photomorphogenesis under low-fluence light. No data on the function of cry2 are available in plants, apart from Arabidopsis (Arabidopsis thaliana). Expression of the tomato (Solanum lycopersicum) CRY2 gene was altered through a combination of transgenic overexpression and virus-induced gene silencing. Tomato CRY2 overexpressors show phenotypes similar to but distinct from their Arabidopsis counterparts (hypocotyl and internode shortening under both low- and high-fluence blue light), but also several novel ones, including a high-pigment phenotype, resulting in overproduction of anthocyanins and chlorophyll in leaves and of flavonoids and lycopene in fruits. The accumulation of lycopene in fruits is accompanied by the decreased expression of lycopene beta-cyclase genes. CRY2 overexpression causes an unexpected delay in flowering, observed under both short- and long-day conditions, and an increased outgrowth of axillary branches. Virus-induced gene silencing of CRY2 results in a reversion of leaf anthocyanin accumulation, of internode shortening, and of late flowering in CRY2-overexpressing plants, whereas in wild-type plants it causes a minor internode elongation.