Temporal and Spatial Regulation of Cyanogenesis in Plants

玫瑰色核果(Prunus spp.)是许多重要的商业作物之一，它们通过内源性酶从生氰苷中释放氰化氢，导致人和牲畜的氰化物中毒。为了更好地了解植物氰化物发生的生化和生理机制，本研究采用生化、免疫细胞化学和分子生物学方法研究了氰基糖苷(R)-核糖苷和(R)-苦杏仁苷在黑樱桃(Prunus Serotina)体内的代谢。在胚胎发育过程中，将测定杏仁苷、杏仁苷、两种生物合成酶(UDPG：扁桃腈葡萄糖基转移酶(GT I)和UDPG：普鲁纳辛葡萄糖基转移酶(GT II)，以及三种分解代谢酶(苦杏仁苷水解酶(AH)、杏仁苷水解酶(PH)和扁桃腈裂解酶(MDL))的水平。GT I和GT II以及苦杏仁苷在种子中的分布将分别通过免疫细胞化学和FT-IR显微光谱来确定。利用前面提出的多克隆抗体，将筛选现有的黑樱桃cDNA文库，以获得AH、PH和MDL的cDNA克隆。这些克隆将被用来通过Northern印迹分析和原位杂交技术开始检测这些基因在胚胎发育过程中的时空表达。发芽幼苗是否利用苦杏仁苷作为氮源，将通过测量上述氰基糖苷和酶以及HCN解毒酶β-氰丙氨酸合成酶在幼苗不同部位的水平和定位来检验。

Rosaceous stone fruits (Prunus spp.) are among many commercially important crops causing cyanide poisoning in humans and livestock through their release of hydrogen cyanide from cyanogenic glycosides by endogenous enzymes. To understand better the biochemistry and physiology of plant cyanogenesis, the metobolism of the cyanoglycosides (R)-prunasin and (R)-amygdalin will be investigated in black cherry (Prunus serotina) using biochemical, immunocytochemical and molecular approaches. Levels of prunasin, amygdalin, two biosynthetic enzymes (UDPG:mandelonitrile glucosyltransferase (GT I) and UDPG:prunasin glucosyltransferase (GT II), and three catabolic enzymes (amygdalin hydrolase (AH), prunasin hydrolase (PH) and mandelonitrile lyase (MDL)) will be assayed during embryogenesis. The distribution of GT I and II and of amygdalin within seeds will be determined by immunocytochemistry and FT-IR microspectroscopy, respectively. Using previously raised polyclonal antibodies, an existing black cherry cDNA library will be screened to obtain AH, PH and MDL cDNA clones. These clones will be utilized to begin examining the temporal and spatial expression of these genes during embryogenesis by Northern blot analysis and in situ hybridization techniques. Whether germinating seedlings utilize amygdalin as a nitrogen source will be examined by measuring the levels and localization of the aforementioned cyanoglycosides and enzymes, along with the HCN detoxification enzyme Beta-cyanoalanine synthase, in various seedling parts.