论文标题:花生白藜芦醇合酶基因表达载体构建及黄瓜遗传转化体系的初步研究
Studies on Construction of Expression Vector with Resveratrol Synthase Gene from Peanut and Genetic Transformation System of Cucumber
论文作者 林建丽
论文导师 陈由强；朱锦懋,论文学位 硕士,论文专业 植物学
论文单位 福建师范大学,点击次数 130,论文页数 74页File Size5910k
2004-04-01论文网 http://www.lw23.com/lunwen_664045967/ 白藜芦醇合酶基因,构建,表达载体,黄瓜,再生体系,转化
resveratrol synthase gene, expression vector, cucumber, regeneration system nthase gene.
芪合酶(STS)基因分为两类：白藜芦醇合酶(RS)基因、银松素合酶(PS)基因。本文阐述了RS基因、PS基因研究进展，特别对落花生属、葡萄属、松属等植物克隆的芪合酶基因数目、结构、同源性进行比较和分类，分析芪合酶基因的表达、调控、诱导特性；对芪合酶的转基因烟草、水稻、小麦、苜蓿等特征、效应进行讨论。 为了对白藜芦醇合酶功能进一步研究，本实验将构建转化双子叶植物的表达载体。研究中使用的花生RS基因已克隆在载体pUCRSH1与pUCRSH2中，分别以pBI与pBI121为表达载体，构建了两个含RS基因的植物表达载体。分别选择合适的限制性内切酶，对表达载体和含RS基因的克隆载体进行酶切，回收片段并纯化，在T4 DNA连接酶的作用下，进行连接、转化，得到重组菌。通过PCR，酶切和序列测定等方式对重组质粒进行检测。构建两个重组表达载体为pBI-RS、pBI121-RS。 提取重组质粒，用冻融法把重组质粒转入农杆菌LBA4404的感受态细胞中，经过筛选培养，用mini-Ti质粒提取方法提取农杆菌质粒，经过PCR检测，确定含RS基因的重组表达质粒已经转入农杆菌中。 通过黄瓜离体再生体系的研究，对可能的影响因素：黄瓜的消毒、外植体选择、生长调节剂、不定芽的分化和抗性筛选等方面进行探索，建立了高效、便捷的离体再生体系。用含重组质粒pBI-RS、pBI121-RS的农杆菌LBA4404侵染双子叶植物黄瓜的外植体，进行黄瓜转基因的初步研究。
Stilbene synthase(STS)gene copied mostly from Arachis, Vitis, Pinus, includes resveratrol synthase (RS)gene and pinosylvin synthase(PS) gene. Progresses in the gene were summarized in this paper, and emphasis is the number, the structure and the homology of the gene. Inducing expression and regulatory of stilbene synthase gene were analyzed. And the resistance of transgenic plants, as tobacco, rice, wheat and alfalfa, were discussed.To further study the function of Resveratrol Synthase , RS gene was transferred to cucumber in this experiment. The RS gene has been already cloned in vectors pUCRSHl and pUCRSH2 before. pBI and pBI121 were used as expression vectors. Then the expression vectors and cloning vectors containing RS gene were digested by the suitable restriction enzymes, the fragments of interest were purified and ligated to each other by T4 DNA ligase. The construct was then transformed into a host strain and screened under the selective pressure of Kanamycin. It was confirmed that the recombinant expression plasmid pBI-RS pBI121-RS has been constructed successfully by examining them with PCR and restriction enzyme digestion.The recombinant expression plasmid was extracted and introduced to Agrobacterium tumefaciens strain LBA4404 with the method of freeze-melt. After culturing and screening the right transformants, the plasmid was extracted with methods of mini-Ti plasmid extracting. It was confirmed with PCR that the recombinant expression plasmidcontaining RS gene has been successfully introduced into Agrobacterium tumefaciens strain LBA4404.There are several factors that can affect the regeneration of adventitious bud: disinfection of cucumber seeds, selection of explant, hormone, differentiation of adventitious bud and resistance screening etc. The efficient protocol for transforming cucumber mediated by Agrobacterium tumefaciens has been established. The explant of cucumber was infected by Agrobacterium tumefaciens strain LBA4404 which contained pBI-RS, pBI121-RS plasmid and the resistance plants was obtained.
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