Cloning And Analysis Of Partial Fragment Of DAD-1 Homologue Gene From Osmanthus Fragrans

Osmanthus fragrans is one of the top ten China Famous Traditional flowers, and for its evergreen and incense advantages, it is the precious ornamental landscape plants. However, it’s short flowering with aging quickly, which impacted its viewing and economic value. In this study, total RNA was successfully isolated from the Osmanthus fragrans ’Liuye Jingui’ petals by improved Trizol method, partial fragment of Actin and DAD-1 （defender against apoptotic death 1） was isolated, and analyzed the expression pattern during the flowering period, in order to provide a theoretical basis for the further molecular mechanism research on aging. Theoretical basis was providing a certain theoretical and practical basis on extending the flowering and viewing period of Osmanthus, increasing the edible, medicinal and fragrance value of sweet Osmanthus. The main results:1. Three methods of isolated total RNA were compared by extracting RNA from Osmanthus petals: CTAB method, traditionary Trizol method and improved Trizol method, the results shown that improved Trizol method got distinct 28S rRNA and 18S rRNA bands, indicating good quality of total RNA. UV absorption of RNA showed a uniform peak at 257 nm without any shoulder peaks. The ratios of the optical density A₂₆₀/A₂₃₀ and A₂₆₀/ A₂₈₀ were about 1.9 and 1.7, respectively, suggesting little contamination of polysaccharides and proteins. The good quality of total RNA was further demonstrated by suitable for downstream molecular works.2. 180 bp housekeeping Actin gene of Osmanthus was cloned by RT-PCR, analysis of the deduced protein sequence of partial Actin gene showed that it exhibited 96% identity to other plants, the results indicated that it was conserved among other plant species.3. In this study, 171 bp fragment of Osmanthus DAD-1 gene was isolated by homologue-sequence-based approach, analysis of the deduced protein sequence of partial DAD-1 gene showed that it exhibited 96 % identity to other plants, the results suggested that it was highly conserved among other plant species.4. We have investigated the temporal and spatial expression pattern of DAD-1 gene during different flowering periods by Real Time PCR way. The results showed that DAD-1 highest expression in the bud, and a rapid decline at the early flowering and full flowering stage, and then slow increased in the full bloom and wilting, however, its expression remained at low level.