【作者】 徐伟荣；

【导师】 王跃进；

【作者基本信息】 西北农林科技大学 ， 果树学， 2005， 硕士

【摘要】 白藜芦醇作为葡萄属植物的抗逆代谢物质-植保素(phytoalexin),是葡萄属植物对病原菌或其它各种诱发因子胁迫下迅速在细胞中累积的一种次生代谢物质,它在细胞中的积累可以提高植物的抗病性。本研究是在获得中国葡萄属野生种华东葡萄白河-35-1芪合成酶基因(STS)的基础上构建中国葡萄属野生种芪合成酶基因植物表达载体,并将其转入根癌农杆菌GV3101 中,然后通过农杆菌介导法将STS 基因导入烟草,借以初步探讨芪合成酶基因在烟草中的转化和表达情况,为其今后在葡萄属植物上的应用提供依据。主要研究结果如下:　1.　中国葡萄属野生种芪合成酶基因的克隆、序列测定　根据本课题组获得的中国葡萄属野生种华东葡萄白河-35-1 芪合成基因cDNA 全长序列,设计特异引物,并进行了修饰和改造,即增加了与植物表达载体pWR306 相匹配的限制性内切酶酶切位点。通过PCR 扩增出目的片段,回收目标产物,克隆到pGEM-Teasy 载体中,得到重组质粒pGEM-T-STS,转化大肠肝菌DH5a,小量提取质粒进行酶切验证。经序列测定,该目的片段为1179bp,与本课题组克隆的芪合成酶基因cDNA 全长序列一致。　2.　植物中间表达载体的构建及鉴定　利用DNA 重组技术,将质粒pSB166 中包含ED35s-O-MCS-UTT-TNOS 的一段核苷酸序列定向克隆到质粒pCAMBIA1303 上,经酶切、电泳鉴定表明,已成功构建了植物中间表达载体pWR306。　3.　中国葡萄属野生种芪合成酶基因植物表达载体的构建　双酶切重组质粒pGEM-T-STS 及表达载体pWR306,将STS 基因片段与线性表达载体pWR306 进行定向连接,构建了芪合成酶基因的植物表达载体pWR-STS,通过酶切、电泳鉴定,STS 基因已成功构建到植物表达质粒pWR306 中,将此克隆命名为pWR-STS。　4.　重组质粒pWR-STS 导入根癌农杆菌GV3101　采用改进冻融法,将重组质粒pWR-STS 导入农杆菌GV3101 中,在卡那霉素和庆大霉素的平板上筛选阳性克隆,至有单菌落出现,进行菌落直接PCR 和提取质粒、PCR 扩增检测,结果表明重组质粒已导入农杆菌GV3101 中。　5.　烟草遗传转化及检测　通过对影响农杆菌介导烟草遗传转化的几个主要因素的探讨,确定了各主要影响因素的值:即取烟草叶片在分化培养基上预培养2天,农杆菌侵染适宜浓度为0.5(OD600),侵染时间6min,在含100　μM 乙酰丁香酮(Acetosyringone,AS)的共培养培养基中暗培养　2天,可以明显提高烟草的转化率。确定了潮霉素选择压力为25mg/L时,可有效地抑制非更多还原

【Abstract】 Resveratrol, a major stress metabolite (phytoalexin) in grapevine, is a kind of secondary metabolites accumulated in cells in response to pathogen stress or other various elicitors. The accumulation of resveratrol in plants can greatly improve plant disease resistance. In this paper, with STS gen cloned, the recombinant plasmid pWR-STS carring STS gene in wild grape species in China was constructed, and introduced into Agrobacterium tumefaciens strain GV3101. Subsequently STS gene was transformed to tobacco by Agrobacterium-mediated method. We initially discussed the transformation and expression of STS gene through studying tobacco genetic transformation, which will provide some beneficial foundation in grape in the future. The results were as following: 1. The cloning,sequencing and analysis of STS gene came from wild grape species in China According to our obtained sequence of STS gene full-length cDNA from Vitis pesudoreticulata Baihe35-1, two specific primers were designed, then were made some modification, adding restriction enzyme sites to match plant middle expression vector. The PCR products were called back and cloned into pGEM-TEasy vector, then recombinant plasmid pGEM-T-STS was constructed. By enzyme digest analysis, the result showed the plasmid containing STS gene was transformed into E.coli. After DNA sequencing, it showed target fragment was 1179 bp in length, which was correspond to our obtained sequence. 2. Construction and identification of plant intermediate expression vector With the application of DNA recombinant technology, the nucleotide sequence of plasmid pSB166 containing ED35s-O-MCS-UTT-TNOS was specifically cloned into plasmid pCAMBIA1303, then plant intermediate expression vector pWR306 was successfully constructed by enzyme digestion and electrophoresis. 3. Construction of plant expression vector carrying STS gene from wild grape species in China After plasmid pGEM-T-STS and plant expression vector pWR306 were double-digested by HindIII and EcoRI, the target fragments were collected and purified respectively, after being ligated, recombinant plasmid pWR-STS was constructed. 4. Introduction of plasmid pWR-STS into Agrobacterium tumefaciens The recombinant plasmid pWR-STS was intruoduced into Agrobacterium tumefaciens strain GV3101 by means of improved freezing-thawing. By screening in the medium containing kanamycin and Gentamycin, and clonoy PCR identification, it was confirmed the result was successful. 5. Study on tobacco genetic transformation by Agrobacterium-mediated system and detection of transformed explants Factors affecting the transformation frequency were studied, and some parameters were determinated as following description. Tobacco leaf explants had been pre-cultured for 2 days; inoculated with active Agrobacterium suspension (absorbance at 600nm reached 0.5) for 5 minutes; co-cultured for2 days on culture medium appended with 100μM AS. The sensitivity of tobacco’s leaf explant to HygB, was used to 25 mg/L,which could effectively inhibit the growth of untransformed tobacco explants. With green fluorescent protein (GFP) detection system, transformed and untransformed explants were separated and identificated in advance, then PCR assay showed that the STS gene had been integrated into tobacco genome.更多还原