Regulation of tryptophan decarboxylase genes from Camptotheca acuminata in transgenic Nicotiana tabacum and Arabidopsis thaliana

Due to the character of the original source materials and the nature of batch digitization, quality control issues may be present in this document. Please report any quality issues you encounter to digital@library.tamu.edu, referencing the URI of the item.Includes bibliographical references (leaves 52-58).Issued also on microfiche from Lange Micrographics.Camptothecin, an increasingly important medicinal compound, is produced by Camptotheca acuminata, the Chinese Happy Tree. This secondary metabolite is a powerful anti-cancer, anti-retroviral compound. With continued research and FDA approval, demand for camptothecin will increase as the source becomes limited. To increase the amount of camptothecin harvested per plant, the camptothecin biosynthetic pathway might be altered by genetic engineering. In order to do this, more information must be gathered about the pathway. One of the key enzymes in the camptothecin pathway is tryptophan decarboxylase (TDC). The TDC enzyme converts tryptophan into tryptamine thereby linking primary and secondary metabolism. In Camptotheca acuminata, TDC is a gene family comprised of one gene (tdc1) which is developmentally regulated, and another (tdc2) which is only elicitable. The regions of the tdc genes that regulate developmental, temporal, and spatial expression have not yet been defined. In the present study a tdc1 genomic clone was isolated and sequenced. The tdc1 gene contained a 23 bp G-C rich region between the translational start site and the putative TATA box of the tdc1 promoter. tdc1::GUS translational fusions between the tdc1 promoter and β-glucuronidase demonstrated that this G-C rich region is important for correct tdc1 expression. Promoter-deletion studies of tdc1 fused to GUS further showed that a negative regulatory element may be present more than 2 Kb upstream of the tdc1 translational start site. This study also explored the regulation of the tdc2 promoter and 3'UTR in translational GUS gene-fusion constructs. Differences between tdc2 expression in C. acuminata, and the results of the gene-fusion constructs in tobacco and Arabidopsis suggest either that the regulation of tdc2 may not lie in the first 1.5 Kb of promoter or in the 240 bp of 3' untranslated region or that these heterologous species lack equivalent transcriptional factors for C. acuminata-like regulation