|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1993 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1992 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Fern antheridiogens are divided into several groups based on cross-testing of biological activities. However, all the antheridiogens characterized so far were derived from Schizaeaceous ferns, and they are gibberellin-related compounds. The purpose of this study is to identify antheridiogens in three species of Scizaeaceous ferns (Lygodium flexuosum, L.circinnatum and Mohria caffrorum) and Ceratopteris richardii. The antheridiogens of C.richardii didn't show any cross-activity on Schizaeaceous ferns, but their biosynthesis was inhibited by treatment with gibberellin biosynthetic inhibitors. From culture media of prothallia of L.flexuosum and L.circinnatum, gibberellin A_<73> methyl ester (GA_<73>-Me) was identified by GC-MS as the principal antheridiogen. From theculture medium of prothallia of Mohria caffrorum, antheridic acid and 3-epi-GA_<63> were similarly identified as antheridiogens. The antheridiogens of C.richardii were shown to consist of at least two compounds, one of them being purified by HPLC to show a single peak on the chromatogrom.
As a case study to investigate mode of action of the antheridiogens, cloning of GA_<73>-Me-inducing genes in L.japonicum prothallia was attempted. Differential screening on a cDNA library from GA_<73>-Me-treated prothallia was carried out using ^<32>P-labeled probes prepared from mRNA from GA_<73>-Me-treated and non-treated prothallia. However, no GA_<73>-Me-inducing gene has been obtained so far. We will attempt subtraction methods to clone antheridiogen-inducing genes.