Abstract:

Matrix attachment regions (MARs) were used to increase resveratrol production in stilbene synthase (STS) gene-transformed tobacco (Nicotiana tabacum L.) in this study. MARs are DNA sequences that bind to the cell's pertinacious nuclear matrix to form DNA loop domains. A range of effects of MARs sequence on mean expression level and variation in expression of transgenes has been reported usually using b-glucuronidase (GUS) gene as reporter gene. The present study investigated the effects of MARs sequence from yeast on transgene expression of STS for the first time. The results of in vitro binding assay showed that the MARs sequence could specifically bind to the matrix of tobacco prepared by lihium diiodosalicylate (LIS) procedure. The stilbene synthase is the key enzyme in metabolic pathway of resveratrol biosynthesis and a cDNA for STS was cloned from Parthenocissus henryana (Hemsl.) Diels et Gilg by RT-PCR method with primers designed according to the conserved sequence of STS gene in grapevine. The cloned sequence showed high nucleotide identity (93.8%) with the STS gene of Vitis vinifera cv. Optima, and the predicted protein sequence also had Cys¹⁶⁴ activity core and IPNSAGAIAGN motifs which were specific for stilbene synthases. With or without flanking MARs from yeast, the STS gene under control of CaMV35S promoter with Ω enhancer has been transferred into tobacco. Northern blot and HPLC analysis of the leaf extracts of transgenic tobacco showed the resveratrol had been produced in STS gene-transformed plants, while there was no resveratrol found in the untransformed tobacco. In stably trans-formed strains, the content of resveratrol in the plants with STS gene flanking by MARs averaged about 2-fold higher than those lacking MARs. The results proved that flanking STS by MARs was one of the effective way to increase resveratrol production in transgenic plants, and this work could also play basic roles on the STS gene transformed vegetables or fruits with strong resistance to disease and benefits to human health.

Key words: matrix attachment regions (MARs), stilbene synthase gene (STS), resveratrol, Parthenocissus henryana , transgenic tobacco, yeast, HPLC