Temporal and Spatial Regulation of Cyanogenesis in Plants

蔷薇核果（Prunus spp.）是许多重要的商业作物之一，通过内源性酶从氰苷中释放氰化氢，导致人类和牲畜氰化物中毒。为了更好地了解植物青花发生的生物化学和生理学，本文将采用生化、免疫细胞化学和分子化学方法研究黑樱桃（Prunus sertina）中青花苷(R)-prunasin和(R)-amygdalin的代谢。在胚胎发生期间，将检测prunasin、amygdalin、两种生物合成酶（UDPG：杏仁腈葡萄糖基转移酶（GT I）和UDPG:prunasin葡萄糖基转移酶（GT II））和三种分解代谢酶（amygdalin水解酶（AH）、prunasin水解酶（PH）和杏仁腈裂解酶（MDL））的水平。利用免疫细胞化学和红外光谱分别测定gt1和gt2以及苦杏仁苷在种子内的分布。利用先前募集的多克隆抗体，筛选现有黑樱桃cDNA文库，获得AH、PH和MDL cDNA克隆。这些克隆将用于通过Northern blot分析和原位杂交技术开始检查这些基因在胚胎发生过程中的时空表达。发芽幼苗是否利用苦杏仁苷作为氮源，将通过测量上述氰苷和酶的水平和定位，以及HCN解毒酶-氰丙氨酸合成酶，在幼苗的各个部位进行检测。

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.