The role of phospholipid lipids signal in rice defense reaction and seeds maturation

过氧化氢（H_2O_2）（0.01 ~ 0.1 mM）诱导悬浮培养水稻细胞中磷脂酸（PA）的快速瞬时积累。在1-丁醇（0.1%）存在的条件下，用H_2O_2处理大米细胞提取液，测定其磷脂酶活性，观察到快速、瞬时的磷脂酰丁醇（PtdBut）生成。h_2o_2活化的磷脂酶为磷脂酶D （PLD）。此外，H_2O_2直接诱导细胞质部分的PLD体外活化，而不处理H_2O_2。lavendustin A （0.05 mM）是一种有效的蛋白酪氨酸激酶（PTK）抑制剂，在体外和体内完全抑制PLD的激活。n -乙酰壳寡糖激发子诱导的植物抗毒素合成在H_2O_2 （0.01 ~ 0.1 mM）存在下增强，而在铁(O_<2^->、1-丁醇（0.1%）和薰衣草素a （0.05 mM）存在下抑制。这些结果表明，h_2o_2诱导的PLD激活增强了水稻细胞中导致植物抗毒素合成的信号转导水稻种子形成过程中的磷脂代谢。在1-丁醇（0.1%）的存在下，测定开花后0-30天水稻种子细胞提取物（DAF）中PLD的活性，至少观察到两种PLD活性。通过RT-PCR对水稻种子0 ~ 20 DAF时制备的RNA进行PLD基因表达谱分析，发现4种PLD基因均有表达。将这4个基因的RNA干扰载体构建成PZH2Bik载体，用农杆菌EHA105菌株转化水稻未成熟胚。然后种植转基因水稻植株，现在正在进行植株的表型分析。

1 Phospholipid metabolism in rice defense reactionHydrogen peroxide (H_2O_2)(0.01-0.1 mM) induced rapid and transient accumulation of phosphatidic acid (PA) in suspension-cultured rice cells. When phospholipase activity in the cellular extract fraction prepared from rice cells treated with H_2O_2 was assayed in the presence of 1-butanol (0.1 %), rapid and transient phosphatidylbutanol (PtdBut) formation was observed. Thus, the H_2O_2-activated phospholipase was concluded to be phospholipase D (PLD). Furthermore, H_2O_2 directly induced in vitro PLD activation in the cytosolic fraction without H_2O_2 treatment. In vitro and in vivo activation of PLD were completely suppressed in the presence of lavendustin A (0.05 mM), a potent inhibitor of protein tyrosine kinase (PTK). Phytoalexin biosynthesis induced by N-acetylchitooligosaccharide elicitor was enhanced in the presence of H_2O_2 (0.01-0.1 mM), whereas it was suppressed in the presence of tiron, a potent scavenger of O_<2^->, 1-butanol (0.1 %), and lavendustin A (0.05 mM). These results indicate that H_2O_2-inducible PLD activation enhances in signal transduction leading to phytoalexin biosynthesis in rice cells.2 Phospholipid metabolism in rice seed formation.When PLD activity in the cellular extract fraction prepared from rice seeds at 0-30 day after flowering (DAF) was assayed in the presence of 1-butanol (0.1 %), at least two kinds of PLD activities was observed. Expression pattern analysis of PLD genes by RT-PCR analysis in RNA prepared from rice seed at 0-20 DAF indicated the expressions of four kinds of PLD genes. The RNA interference vectors of the four genes were constructed into vector PZH2Bik and transformed into immature rice embryos used with Agrobacterium tumefacience strain EHA105. Then the transgenic rice plants were grown and the phenotype analysis of the plants is now underway.