【作者】 谭琳；

【导师】 郑学勤；

【作者基本信息】 华南热带农业大学 ， 作物遗传育种， 2002， 博士

【摘要】 白藜芦醇（resveratrol）是由一些种子植物对受伤作出反应时产生的一种植保素，实验证明白藜芦醇能够调节脂代谢和抑制低脂蛋白的氧化和血小板的凝集，而且能够提供心血管保护，并具备抗炎症和抗癌特性，对保护人体健康十分有益。番茄作为一种大众化、日常食用蔬菜，富含维生素A、B和P，特别是其番茄红素（lyxopene）对预防前列腺癌具有一定的作用，因此，将合成白藜芦醇的芪合酶基因导入番茄，获得更具保健价值的番茄新品种将产生重要的社会和经济效益。 本研究包括四个方面的内容：芪合酶基因（Stilbene synthase gene）的克隆与全序列分析；芪合酶基因（stilbene synthase gene）植物表达载体的构建；农杆菌LBA4404介导的番茄遗传转化：HPLC技术分析转基因植株中表达白藜芦醇的含量。 通过PCR扩增和克隆，获得了来自葡萄（vitis vinefera）品种雷司令和粉红玫瑰叶片中的芪合酶基因（Stilbene synthase gene）。DNA序列分析结果表明，来自雷司令的芪合酶基因全长1617bp，与文献报道的该基因（pVIN）比较，碱基同源率为99％，来源于粉红玫瑰的芪合酶基因全长1622bp，与pVIN的序列比较，碱基同源率为98％。两者都编码392个氨基酸与pVIN一致。 用BamH I和Sac I同时酶切pS2（S2表示来自雷司令的芪合酶基因）、pS1（S1表示来自粉红玫瑰的芪合酶基因）以及pBI121、pEV2，使得S2、S1分别插入替代pBI121、pEV2中的GUS基因，构建成植物表达载体pBS2、pEV2S1，pBS2中含CaMv35s组成型启动子，使S2基因能在番茄植株的各个部位表达；pEV2S1则含有果实特异性启动子TFP2，使S1基因只在番茄果实中表达。经双酶切鉴定，证实S2、S1基因己插入到pBS2和pEV2S1中。将pBS2和pEV2S1通过三亲交配法导入农杆菌，经PCR鉴定证实pBS2和pEV2S1已导入LBA4404。 番茄TX0014和抗青枯3两个品种对kan的敏感程度差异不大，当kan浓度为50mg／L时，叶盘黄化，几乎不形成愈伤：而50mg/L的kan使番茄植株不能生根和生长。通过对转化条件进行优化，发现在含有100μmol/L AS的MS培养基中，用0D₆₀₀值为0.1的新鲜活化的LBA4404侵染2-3分钟后子叶叶盘在kan50mg/L的MS培养基上的出愈率和分化率最佳。 通过kan生根筛选，获得转芪合酶基因（S2）的番茄TX0014品种5株，提取这5株转基因植株和一株对照植株的总DNA，作PCR检测，5株均呈阳性，而对照植株无特异片段出现，继续Southern检测，结果表明，芪合酶基因（S2）已整合到其中3株的基因组中。取这3株的嫩叶，对照植株（2株）的嫩叶，作HPLC分析，3株转基因植株中均检测到白藜芦醇的存在。其含量分别为12.45mg／kg，5.35mg／kg，4.35mg／kg，而对照植株未检出，这表明芪合酶基因（S2）已在转基因番茄中表达。 本研究，为番茄的品质改良育种，探索出一条新的方法和途径，具有一定的理论意义和实际应用前景。更多还原

【Abstract】 Resveratrol(trans-3,5,4 -trihydroxystilbene) is a naturally occurring phytoalexin produced by some spermatophytes,such as grapevines,in response to injury. Research shows that resveratrol possesses activities of modulation of lipid metabolism,inhibition of lipid peroxidation(LDL,membranes) and platelet aggregation,etc. Meanwhile as it can reduce coronary heart-disease mortality and features the activity of anti-inflammatory,it is condidered to have some beneficial effects on human health. As a popular vegetable,tomato is rich in vatamin A,B,P and other nutrients;among them,lyxopene can prevent prostate gland tumor. So introducing stilbene synthase gene into tomato to get new health-protection tomato will have important social and economic effect.Our research is made up of four parts;the cloning and sequencing of stilbene synthase;tomato genetic transforming mediated by Agrobacterium-LBA4404;quantification of resveratrol in transgenic tomato by HPLC analysis.Stilbene synthase genes(l,2)from Vitis venefera were cloned and sequenced. The results of sequence analysis showed the total length of S2 is 1617bp,the homology between S2 and pVIN(reported) is 99%;the total length of SI is 1622bp,the homology between SI and pVIN(reported) is 98%. Deduced amino acid sequence of S1,S2,pVIN were also highly homologous each other(98%,99% in each case). The stilbene synthase genes were excised from the plasmids by BamH I and Sac I digestion and intergrated into a binary vector,pBI121 and pEV2,from which the P -glucuronidase(GUS) gene sequence had been removed by the same digestion to prepare a 35s promoter-stilbene synthase 2-nopaline synthase polyadenylation site construct and a TFP2 promoter-stilbene synthase 1-nopaline synthase polyadenylation site construct. The recombinant plasmids were called pBS2,pEV2S 1 .respectively.There was no evident difference between the cultures of variety TX0014 and anti-Pseudomonas 3 for their sensitivity to Kanamycin(Kan). The leaf discs turned yellow and calli were hardly induced at 50mg/L Kan concentration in the medium M1. Kanamycin at 50mg/L concentration suppressed the root growth and the plantgrowth The efficiency of callus induction and regeneration on the medium Ml containing 50mg/L Kan was the highest when the leaf discs were infected with Agrobacterium tumefaciens containing lOOmol/L AS between 2 and 3 minutes.Five plants (TX0014) regenetated from selection medium were obtained. The .presence of the stilbenesynthase gene 2 in the regenerated plants was checked by PCR . All of the plants showed a clear band corresponding to the relevant sequence of the gene,but the control showed no band. Southern blot analysis suggest that S2 gene has been integrated in the genome of three PCR-positive regenerated plants. The rest two regenerated plants did not show a band corresponding to S2. A HPLC analysis of leaf extract from 3 transformant and 2 non-transformant was made. It indicates that resveratrol was produced in these three transformants at a concentration of 12.5ug/g,4.55ug/g,5.35 ng/g respectively,the resverarol production was not examined in non-transformants.Our research can lay a good foundation for tomato quality breeding,meanwhile the tomato containing resveratrol is also potential for providing protection on human health.更多还原