An active Catharanthus roseus desacetoxyvindoline-4-hydroxylase-like gene and its transcriptional regulatory profile.

Chen Zhou,Jin Zhang,Zhi-Bi Hu,Shu-Juan Zhao

doi:10.1186/1999-3110-55-29

Abstract

BackgroundDesacetoxyvindoline-4-hydroxylase is a key enzyme in the biosynthesis of vindoline, the important intermediate leading to vinblastine and vincristine in Catharanthus roseus.ResultsA d4h-like gene has been isolated from C. roseus C20hi cells based on an EST sequence from the Suppression Subtractive Hybridization cDNA library. The full length cDNA of d4h-like was 1427 bp encoding 372 amino acids. It had 66% identities and 80% positives with d4h at the amino acid level. It belonged to 2-oxoglutarate dependent oxygenase superfamily as d4h did. Real-time quantitative PCR analysis revealed that d4h-like was expressed high in roots, flowers and C20hi cells, very low in leaves and stems. Methyl jasmonate could significantly increase the accumulation of d4h-like transcripts. 2,4-D inhibited its expression. An approximate 2,910 bp of 5′-promoter region of d4h-like was obtained, fused to GUS reporter gene and analyzed with fluorescence quantitative assays using transient expression in C. roseus cell suspensions, indicating that d4h-like promoter could drive GUS gene expression in vivo.ConclusionThese results suggest that d4h-like is closely related with d4h in the genetic evolution but with different transcriptional expression profiles. It may be revolved in the hormone-independency of C20hi cells.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-55-29) contains supplementary material, which is available to authorized users.

Highlights

Desacetoxyvindoline-4-hydroxylase is a key enzyme in the biosynthesis of vindoline, the important intermediate leading to vinblastine and vincristine in Catharanthus roseus
The ORF of d4h-like was fused with the 33 amino acid residues in the pRSET vector at the N-terminal
After induced by IPTG, an approximate 44KD of specific watersoluble protein was expressed in E. coli BL21 (Figure 2), which was right the predicted molecular weight of the recombinant D4H-LIKE protein

Summary

Introduction

Desacetoxyvindoline-4-hydroxylase is a key enzyme in the biosynthesis of vindoline, the important intermediate leading to vinblastine and vincristine in Catharanthus roseus. Vinblastine and vincristine are powerful anticancer compounds produced by Madagascar periwinkle (Catharanthus roseus (L.) G. D4H was a 2-oxoglutarate-dependent dioxygenase and could be induced by light and red-light (Verpoorte et al 2000). It was localized at cytoplasm and its mRNA were associated with the laticifer and idioblast cells of the leaves, stems and flower buds (St-Pierre et al 1999). Hydroxylase assays and RNA-blot hybridization studies showed that D4H activity was closely related to the levels of d4h transcripts, occurring predominantly in young leaves and in much lower levels in stems and fruits (Vazquez-Flota et al 1997). The biosynthesis of vindoline was light-induced and had tissue-specific accumulation profile which was coincided with the expression patterns of d4h (El-Sayed and Verpoorte 2007)

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Conclusion