Isolation and characterization of alkaloid biosynthetic genes from Camptotheca acuminata

Abstract

Camptotheca acuminata is the primary source of the mono-indole alkaloid camptothecin (CPT) and its derivative 10-hydroxy camptothecin, which are currently in clinical trials as anticancer drugs, and also have been shown to inhibit the replication of the AIDS virus in low dosage. Two genes involved in the biosynthesis of these compounds, tryptophan decarboxylase (TDC) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), were cloned from C. acuminata and their expression was analysed. TDC catalyzes the conversion of tryptophan to tryptamine, and is an important step early in the biosynthetic pathways for the synthesis of indole alkaloids. A partial 3' genomic clone for the C. acuminata tdc gene was isolated from a genomic DNA library using a Catharanthus roseus tdc cDNA as a heterologous probe. Further 5' sequence was obtained by nested PCR, using internal primers plus a degenerate primer based on comparisons with other known decarboxylases. RNA gel blot analysis indicates that tdc is expressed primarily in the tree bark, where the most camptothecin has been reported. The tdc message is also seen in developing young seedlings and is inducible in leaves by the plant hormone methyl jasmonate (MeJA). HMGR supplies mevalonate for the synthesis of the terpenoid component of CPT, as well as for the formation of many other primary and secondary metabolites. A full-length C. acuminata gene encoding HMGR (hmgl) was isolated using a PCR generated probe. RNA gel blot analysis of plant parts and seedlings shows a strong hmgl signal in two-week old seedlings not detected in older vegetative tissues. Regulation of the hmgl promoter was studied in transgenic tobacco using three translational fusions of the promoter to the β-glucuronidase (GUS) reporter gene. Histochemical analysis of plants containing each of the three promoter fusions showed similar developmental and spatial expression patterns. All three constructs showed induction of GUS activity by wounding, and complete suppression by treatment with MeJA. Gel electromobility shift assays of the smallest promoter fragment identified differential binding activity in proteins from untreated, wounded and MeJA treated plant tissues. A model is proposed to account for these results.