RESUMO
We report the expression profile of acyl-lipid Delta12-desaturase (desA) gene from Synechocystis sp. PCC6803 and its effect on cell membrane lipid composition and cold tolerance in prokaryotic (Escherichia coli) and eukaryotic (Solanum tuberosum) cells. For this purpose, a hybrid of desA and reporter gene encoding thermostable lichenase (licBM3) was constructed and used to transform these cells. The expression of this hybrid gene was measured using qualitative (Petri dish test, electrophoregram and zymogram) and quantitative methods (spectrometry and gas liquid chromatography assays). The maximum level of linoleic acid in the bacterial cells containing hybrid gene was 1.9% of total fatty acids. Cold stress tolerance assays using plant damage index and growth parameters showed that cold tolerance was enhanced in primary transgenic lines because of increased unsaturated fatty acid concentration in their lipids. The greatest content of 18:2 and 18:3 fatty acids in primary transgenic plants was observed for lines 2 (73%) and 3 (41%). Finally, our results showed that desaturase could enhance tolerance to cold stress in potato, and desaturase and lichenase retain their functionality in the structure of the hybrid protein where the enzymatic activity of target gene product was higher than in the case of reporter lichenase gene absence in the construction.
Assuntos
Adaptação Fisiológica , Temperatura Baixa , Células Eucarióticas/metabolismo , Ácidos Graxos Dessaturases/genética , Células Procarióticas/metabolismo , Solanum tuberosum/fisiologia , Synechocystis/enzimologia , Adaptação Fisiológica/genética , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Escherichia coli/metabolismo , Regulação da Expressão Gênica de Plantas , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Ácidos Linoleicos , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Solanum tuberosum/genética , Solanum tuberosum/crescimento & desenvolvimento , Estresse Fisiológico/genética , Synechocystis/genética , Fatores de Tempo , Transformação GenéticaRESUMO
The desC gene for the acyl-lipid Delta9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus was introduced into Nicotiana tabacum under control of the 35S promoter. Expression of the desaturase was confirmed by Western blotting. Lipid analysis revealed that lipid content and the extent of fatty acid unsaturation significantly increased in leaves of transgenic plants. Chilling tolerance of those plants also increased, as estimated by the electrolyte leakage from the tissues damaged by cold treatments. Seeds of plants that expressed the desC gene imbibed at low temperatures demonstrated higher chilling tolerance than those of the control plants. The results demonstrate that the cyanobacterial thermophilic acyl-lipid desaturase was efficiently expressed in tobacco at ambient temperatures, and its expression resulted in the enhanced chilling tolerance of the transgenic plants.
