核基质结合区对转爬山虎芪合酶基因烟草中产生白藜芦醇的作用

采用核基质结合区(MARs)来提高转芪合酶基因(STS)烟草(Nicotianatabacum L.)中白藜芦醇产物的含量.MARs是细胞中能与核基质特异紧密结合的DNA片段,体外结合实验表明克隆自酵母的MARs序列能特异地与烟草核基质结合.芪合酶是白藜芦醇生物合成中的关键酶,用RT-PCR方法从川鄂爬山虎(Parthenocissus henryana(Hemsl.)Diels et Gilg)中克隆了与葡萄芪合酶基因有较高同源性的芪合酶编码区,将其置于CaMV35SΩ强启动子下,分别构建两侧带有MARs及不含MARs序列的表达载体,通过农杆菌介导转化烟草.Northern blot及HPLC等分析表明STS基因已整合至烟草染色体中并正常转录,且表达的外源芪合酶在烟草中可催化其底物合成白藜芦醇产物.与对照相比,MARs的存在使转芪合酶基因烟草中白藜芦醇的含量平均提高了约一倍.MARs在转芪合酶基因植物中的应用也为获得抗病性更强、白藜芦醇含量更高、更保健的转基因果蔬的研究奠定了基础.

Effect of Matrix Attachment Regions on Resveratrol Production in Tobacco with Transgene of Stilbene Synthase from Parthenocissus henryana

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.