25 蛍光ディファレンシャル・ディスプレー法を用いたエンドウ矮性変異株「1kb」の幼苗におけるブラシノライド応答遺伝子の探索

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In the 34 annual meeting, we reported the effect of timing of brassinolide(BL) treatment on a brassinosteroid deficient 1kb mutant of dwarf pea(Pisum sativum, cv Torsdag). In this study, we carried out a large scale screening for BL-regulated genes by using a fluorescent differential display(FDD) technique. The 1kb epicotyls at 29th hour after sowing were treated with BL (1×10^<-7>M) or solvent. Total RNA was prepared from these 1kb epicotyls after 0, 0.5, 1.5 and 4.5 hour treatment with or without BL. mRNA fingerprints of these sample were compared to screen for BL-regulated genes. Approximately 13,000 cDNA bands were displayed by FDD using 160 primer combinations, and then six differentially expressed FDD fragments were detected. Among them, two FDD fragments(CB5, CB12) were isolated and used as probes for screening of full-length cDNA clones. A full-length cDNA clone corresponding to CB5 was encoding a novel cytochrome P-450 related protein which shown 60% similarity with other cytochrome P-450 at protohem binding site. A cDNA clone corresponding to CB12 was characterized by the presence of adjacent 26 amino acid repeats which are contained pea tissue-specific gene S2 and chickpea cell-elongation gene CanST-1. A number of 26 amino acid repeat of isolated clone was different with those of S2 and CanST-1, suggesting that isolated clone is encoding a new member of those proteins.
植物化学調節学会の論文
2000-11-02