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

发展专门的工厂， 环境或维持作物生产力和安全的粮食供应， 在金属污染土壤中生长的农业植物需要 了解植物的金属耐受机制。这 理解将提供可用于筛选植物的性状 通过常规育种策略产生或可用于 用所需的性状改造植物。在这项工作完成之前， 然而，金属诱导基因表达的基础研究必须 进行鉴定基因和基因产物参与金属 宽容我建议系统地研究基因表达， 耐金属植物组织培养和鉴定基因及基因产物 涉及金属耐受性。 差异显示逆转录聚合酶链反应将 可用于研究镉诱导的耐金属植物基因表达 组织培养耐重金属曼陀罗总RNA的提取 在不存在和存在镉的情况下的植物细胞培养将用于 逆转录酶反应，然后通过PCR扩增。的 扩增产物用聚丙烯酰胺凝胶电泳分离， 扩增产物的电泳和并排比较 从不同的样品中检测差异表达的cDNA 片段将这些cDNA片段从凝胶中切下， 扩增、克隆和测序。这些序列将用于搜索 核苷酸和肽序列文库，用于可能鉴定 所述序列代表的基因或蛋白质。确认 差异表达将通过北方分析进行。全长 任何未鉴定的cDNA片段的cDNA将被分离、测序并 鉴定这项工作将提供有关差异的信息- 在D.无害和 让我们能够回答关于金属特异性和植物 种属特异性的信息获得与此建议 研究在未来。

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.