TOXIC METALS IN PLANTS--METAL INDUCED GENE EXPRESSION IN METAL TOLERANT PLANTS

发展专门的植物，以去除有害金属 环境或维持作物产量和安全的粮食供应 在金属污染的土壤中生长的农业植物需要 了解植物对金属耐性的机制。这 理解将提供可用于筛选植物的特征 由传统的育种策略产生的或可用于 设计出具有所需特征的植物。在完成这项工作之前， 然而，金属诱导基因表达的基础研究必须 对金属中涉及的基因和基因产物进行鉴定 宽容。我建议系统地研究基因在一种 耐金属植物组织培养及基因和基因产物鉴定 与金属耐受性有关。 差异显示逆转录聚合酶链式反应 用于研究镉诱导的金属耐性植物的基因表达 组织培养。耐金属曼陀罗总RNA的提取 在无镉和有镉存在的情况下，植物细胞培养将用于 逆转录酶反应后用聚合酶链式反应进行扩增。这个 扩增产物将用聚丙烯酰胺凝胶进行解析 扩增产物的电泳法和比对分析 从不同的样本中检测到差异表达的cDNAs 碎片。这些cdna片段将从凝胶中切下，用于 扩增、克隆和测序。这些序列将用于搜索 核苷酸和多肽序列文库用于可能的鉴定 序列所代表的基因或蛋白质。确认 差异表达将通过Northern分析进行。全长 任何未识别的cdna片段都将被分离、测序和 已确认身份。这项工作将提供关于不同的信息- 表达镉诱导的基因或基因产物 让我们回答有关金属专一性和植物的问题 利用该建议获得的信息的物种特异性 未来的研究。

The development of specialized plants to either remove harmful metals from the environment or to maintain crop productivity and a safe food supply in agricultural plants growing in metal contaminated soils requires an understanding of the mechanisms of metal tolerance in plants. This understanding will provide traits which can be used to screen plants produced by conventional breeding strategies or which can be used to engineer plants with the desired traits. Before this work can be done, however, fundamental studies of metal-induced gene expression must be conducted to identify the genes and gene products involved in metal tolerance. I propose to systematically investigate gene expression in a metal-tolerant plant tissue culture and identify genes and gene products involved in metal tolerance. Differential display reverse transcriptase polymerase chain reaction will be used to study cadmium-induced gene expression in a metal-tolerant plant tissue culture. Total RNA isolated from metal tolerant Datura innoxia plant cell culture in the absence and presence of cadmium will be used for reverse transcriptase reactions followed by amplification by PCR. The products of the amplification will be resolved by polyacrylamide gel electrophoresis and side by side comparisons of the amplification products from the different samples will detect differentially expressed cDNA fragments. These cDNA fragments will be excised from the gels for amplification, cloned and sequenced. The sequences will be used to search nucleotide and peptide sequence libraries for possible identification of the genes or proteins represented by the sequences. Confirmation of differential expression will be done by Northern analysis. Full-length cDNAs for any unidentified cDNA fragments will be isolated, sequenced and identified. This work will provide information about differentially- expressed cadmium-induced genes or gene products in D. innoxia and position us to answer questions about the metal-specificity and plant species-specificity of the information obtained with this proposed research in the future.