【作者】 虢利钢；

【导师】 易自力； 蒋建雄；

【作者基本信息】 湖南农业大学 ， 植物学， 2006， 硕士

【摘要】 纤维素是世界上最丰富、具有巨大商业价值的生物多聚体，几十年来一直是人们研究热点。近年来，随着基因组学的发展，关于纤维素的生物合成及相关基因表达调控的研究取得较大的进展，这对于植物纤维素的生产和应用具有重要意义。本实验室利用一个与拟南芥中的KOR基因（编码跨膜内切-1，4-β-葡聚糖酶）高度同源的棉纤维的表达序列标签（expressed sequence tags，EST），设计特异性引物，通过PCR筛选方法从陆地棉（Gossypium hirsutum L．）纤维cDNA文库中分离出了它的全长序列（GhCEL，GenBank accession number：AY574906）。在此工作基础上，本研究构建了GhCEL基因植物表达载体，导入模式植物烟草中，并分析了转化植株的纤维素含量的变化。 植物表达载体的构建：通过特异引物设计从克隆载体上扩增出目的基因GhCEL全长序列，并引入到中间载体SK-pBlueScript+。从pFGE5941载体中引入CaMV35启动子序列，构建pCAMBIA 1302：CaMV35。将中间载体SK-pBlueScript+：GhCEL中的目的基因片段连接到pCAMBIA 1302：CaMV35上，即构建了GhCEL基因与绿色荧光蛋白基因（GFP）融合的GhCEL基因的植物表达载体。 GhCEL基因转化烟草：利用根癌农杆菌介导的叶盘法转化烟草，将侵染过含有目的基因农杆菌的烟草叶盘置于MS共培养基中，共培养3天后，转置于含潮霉素(25mg·L^-1)和头孢霉素(500mg·L^-1)的MS选择培养基中。同时设未经农杆菌侵染的叶盘置于MS选择培养基中作对照。取叶盘周围产生的愈伤组织，在MS继代培养基上继代培养，待芽长至约3cm时，将芽切下转入MS生根培养基中，同时以未经转化的不定芽作对照。将生根后的幼苗移入土壤中栽培，获得了一批具有潮霉素抗性的转化植株。 取抗性植株叶片，按CTAB法提取烟草基因组DNA，并以其为模板用特异引物进行PCR扩增，同时设未转基因烟草为对照。结果表明，73.3％的抗性植株能扩增出360bp的特异带，而未转化的对照植株未见相应的扩增带。进一步PCR-Southern杂交表明，阳性植株都有明显的阳性杂交信号而阴性对照无杂交信号，证明了GhCEL基因已整合到了烟草基因组中。 对转基因烟草植株纤维素含量测定结果显示：转化植株纤维素平均含量为22.32％，更多还原

【Abstract】 Cellulose has become the worldwide hotspot in biological research field in the past decades due to its great abundance in the nature and its remarkable commercial value in the world. With the development of genomics, recent studies have highlighted the cellulose synthase complex and the role the genes involved in cellulose biosynthesis pathway in plant. The findings will be the guidance of the production and application of cellulose. Previously, according to an expressed sequence tag （EST） of cotton fiber a full-length cDNA was isolated （which is designated GhCEL, GenBank accession number: AY574906） from a fiber cDNA library of Gossypium hirsutum L. by the PCR screening by our laboratory, which is highly homologous to KOR gene of Arabidopsis thaliana. In this research GhCEL expression vector was constructed and transformed into tobacco. We also analysed the change of contents of cellulose of the transgenic plants.1 .Construction of expression vector: The special primer pair was designed to amplify a full-length of GhCEL gene from cloning vector by the PCR reaction. GhCEL gene was inserted into a cloning vector SK-pBlueScript+, and the resulting recombinant vector was named as SK-pBlueScript+: GhCEL. At the same time CaMV35 promoter digested from pFGE5941 vector was inserted into expressing vector pCAMBIA 1302, and the resulting vector was named as pCAMBIA 1302: CaMV35. GhCEL digested from intermediate vector SK-pBlueScript+: GhCEL was inserted into pCAMBIA 1302: CaMV35, and then GhCEL-GFP fusion gene expressing vector was obtained.2.The tobacco transformation: The tobacco leaf discs which were infected by agrobacterium tumefaciens with GhCEL gene was put into MS cocultured media. After 3 days, they were transferred into the selective media with hygromycin (25mg-L^-1) and cefotaxime (500mg-L^-1). The leaf disc without GhCEL gene were transferred into the selective media as the control. The callus around the leaf disc were transferred to the subculture media. When the bud were more than 3 centimeters,they were cut and transferred into MS rooting media and the young rooted buds were planted into soil.更多还原