植物氰丙氨酸合成酶的基因克隆及其抗病相关性表达研究

Cyanide is produced in plants ubiquitously. As cyanide can inhibit mitochondrial electron transport, the processes of cyanide-detoxification and cyanide-resistance play very important roles in substance and energy metabolisms in plant cells, and serve as a base for accomplishing diverse physiological functions and even for responses again pathogens in plants. Cyanoalanine synthase (CAS) is the key enzyme for scavenging cyanide in plants. Cyanide-resistant respiration is specific in plant cells and is insensitive to cyanide. However, there is still very limited knowledge about cyanide-detoxification and cyanide-resistant respiration in plants. In this project, cyanogenesis, cyanide-detoxification and cyanide-resistant respiration, especially CAS, have been studied with tobacco and rice plants as materials. The following meaningful results have been obtained..1). Isolation and purification procedures of tobacco CAS were studied. Precipitation by 0.20-0.60S ammonium sulphate was first used. The precipitated protein was resuspended and was purified by anion exchange chromatography on DEAE Sephacel. Two CAS fractions were obtained. The peak 2 fraction was further purified by hydrophobic chromatography on Octyl Sepharose. The peak 1 fraction was purified by cation exchange chromatography on Sepharose CM. A CAS fraction was eluted. This CAS fraction was further purified by hydrophobic chromatography on Octyl Sepharose.2). The subcellular distribution of tobacco and rice CAS isozymes were determined. It was found that both of the two CAS forms existed in tobacco leaf cytoplasm. Only CASt1 (or CASr1 ) was observed in mitochondria and no CAS existed in chloroplast.3). It was found that the anti-oxidation abilities of the two CAS isozymes of tobacco are quite different. CAS extraction from tobacco using buffer without any reductants, such as Cys, DTT, produced only one CAS band on non-denaturing polyacrylamide gel. But addition of reductant into the extraction buffer resulted in two CAS bands, CASt1 and CASt2.4). A great difference was found between CASs of tobacco and rice. No effect was observed by reductant on CAS isozyme bands on non-denaturing polyacrylamide gel. Furthermore, the positions of the CAS isozyme bands of tobacco and rice were quite different on non-denaturing polyacrylamide gel..5). Part of the CAS cDNAs of tobacco and rice were cloned. 6). Hypersensitive response (HR) with necrotic lesions was observed on tobacco (Nicotiana glutinosa) leaves after inoculation of tomato mosaic virus (ToMV). A drastic increase in cyanide content was found in the tobacco leaves during the HR. This cyanide was probably produced along with ethylene production, as an obvious increase in ethylene production rate was found during the HR. The CAS activity distinctively decreased in the leaves during the HR, suggesting a decrease in cyanide detoxification ability. Treatment with 50 mM KCN solutions caused similar necrotic lesions on tobacco leaves. The above results indicated that during the HR of tobacco (Nicotiana glutinosa) against ToMV cyanogenesis increased, but the cyanide detoxification ability decreased, suggesting endogenous cyanide may take part in the resistance of tobacco against viruses. 7). The tobacco leaves could recover from water deficit for 1 to 2 days. The CAS activity in the leaves increased sharply upon water deficit, but declined after tobacco plants were re-watered. It is inferred that CAS takes part in the resistance of tobacco upper leaves against water deficit stress..8). CAS activities were detectable in rice seeds of Xiushui 11 and Zhenong 982 on the 3rd day since imbibition start, and continuously increased during the following germination process. All the CAS activities in roots, leaves and endosperm of Xiushui 11 seeds increased during germination. It was found that the cyanide content in dried Xiushui 11 rice seeds was quite low, but it increaseed along with the germination process of the seeds. The changing patterns of CAS activity and the cyanide content were quite similar in the seeds durin

从烟草叶片中分离纯化氰丙氨酸合成酶，制备抗体，克隆基因，比较抗／感性烟草和水稻在分别接种烟草花叶病毒和稻瘟病菌前后的内源氰化物水平、氰丙氨酸合成酶湃性及其蛋白与核酸表达水平的差异，以探明植物如何通过调控氰丙氨酸合成酶的基因表达式平利用内源氰化物参与抗病防卫反应。