Functional Analysis of a Senescence-associated Phospholipase A Gene in Oncidium Gower Ramsey

• Main Authors: Pu-Huan Liu, 劉朴桓
• Other Authors: 鄭石通
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/88015766047073015096

碩士 === 國立臺灣大學 === 植物科學研究所 === 96 === OSAG78 is a senescence-associated gene cloned from Oncidium flower and was predicted as a lipid acyl-hydrolase protein. The full-length cDNA of OSAG78 was isolated and its ectopic expression of OSAG78, driven by 35S promoter in Arabidopsis, was analyzed. Compared with wild-type plants, the transgenic plants display smaller body size, stiffer inflorescence stem, thicker leaves, shorter siliques and more round-shaped flowers. Moreover, the 35S::OSAG78 transgenic lines display different flowering time. The mechanism of over-expressing OSAG78 in Arabidopsis is further studied in this research. According to RT-PCR assays, OSAG78 was regulated either during flowing time or after ethylene treatment in Oncidium. We found that OSAG78 showed the different expression pattern comparing to patatin-like proteins in Arabidopsis. Based on the leaf trichome distribution and phenotype of 35S::OSAG78 transgenic lines, the effects of OSAG78 in Arabidopsis were associated with an increase in the length of developmental phases of plant. It was assumed that the growth retardation may caused by GA deficiency or blocking in GA responses. The contents of bioactive GA4 and GA7, quantified by GC-MS, as well as the levels of AtGA20ox1 and AtGA2ox1 transcripts, quantified by real-time PCR, were lower than those in the wild-type plants. These results suggest that OSAG78 accumulation affects GA metabolism through the repression of biosynthetic steps catalyzed by GA 20-oxidase. The content of chlorophyll and the expression of AtSAG in the detached leaves of transgenic and wild-type plants were investigated. In the leaves with or without ethylene, the expression of AtSAG was lower in transgenic lines than in wild-type plant. However, effects in transgenic plant were not observed after ABA treatment, and the application of GA and BAP decreases the expression of AtSAG12 in both transgenic and wild-type plants. Therefore, there should be another pathway to display effects of 35S::OSAG78 transgenic lines. By analysis of protein crude extracts, phospholipase A activity was higher in transgenic lines than in wild-type plants. Furthermore, a recombinant GST:OSAG78 fusion protein that overexpressed in Escherichia coli was accumulated in the insoluable protein pellet displaying lipase and phospholipase A activity. This result agrees with the previous prediction that OSAG78 protein is hydrophobic. To confirm the membrane association of OSAG78, a OSAG78:GFP fusion polypeptide was transiently expressed in onion (Allium cepa) epidermal cells, indicating that OSAG78 is localized at the cytoplasmic membrane. These results point out that over-expression of membrane-associated patatin protein OSAG78 that had phospholipase A activity affected GA biosynthesis in plant.