【作者】 曹克浩；

【导师】 肖兴国；

【作者基本信息】 中国农业大学 ， 生物化学及分子生物学， 2003， 硕士

【摘要】 本项研究分为两个部分：“水稻启动子的序列和功能分析”和“大肠杆菌argE的克隆及对烟草的转化”。 第一部分：对已克隆的启动子Osg6B’进行的序列分析表明，Osg6B’全长1688bp，与报道的Osg6B有98％的同源性，两者在转录起始位点、TATA box及其它花药特异性启动子共有的保守序列TGTGG完全相同。但是与报道的Osg6B比较，在决定时空特异性的0～-1273bp功能区域内，有18处出现18碱基替换，18处出现22碱基缺失，3处出现3碱基插入；在核心功能序列区域（-1095bp～-1273bp）内，有3处出现3碱基替换，6处出现6碱基缺失。在序列分析的基础上，对Os96B’在转基因植株中的时空特异性进行了分析。将克隆的启动子与报告基因GUS相连，构建植物表达载体，通过农杆菌介导转化烟草。T₀代成熟植株中，在花药发育的不同时期和不同部位，以及在转基因植株的萼片、花瓣、柱头、花丝、花粉都检测出GUS活性。在9棵T₀幼苗植株中，2棵检测为阴性，4棵在根、茎、叶中检测出GUS活性，3棵在茎、叶中检测出GUS活性。3／4的T₁代总数的抗性幼苗的根、茎、叶检测到GUS活性。外源基因的遗传分析表明，T₁代的表型分离符合孟德尔法则。研究表明，所克隆的Osg6B’不具有花药特异性，而具有组成型表达的特征。 第二部分：从大肠杆菌E．coli中克隆出argE基因，全长1152bp,推导编码总数383个氨基酸的N-乙酰-鸟氨酸酶（N-acetylornithinase，NAO）。与报道的argE序列相比，DNA序列和推导的氨基酸序列完全相同。将argE与组成型启动子CaMV 35S相连，构建植物表达载体，通过农杆菌介导转化烟草。经过GUS染色、PCR及Southern鉴定获得了转基因植株。argE基因在转基因植株中的功能研究正在进行。更多还原

【Abstract】 This study consisted of two parts: sequence and functional analysis of the cloned promoter Osg6B’ from rice and molecular cloning and transferring into tobacco ofargE from E.coli.In the part I, the sequence of the cloned promoter Osg6B’ was first analyzed. Osg6B’ had a whole length of 1688 bp and 98% homology to known sequence of promoter Osg6B. Its transcriptional start point, TATA boxes and the consensus sequences "TGTGG" conserved usually in anther-specific promoters were identical to those of reported Osg6B. However, the cloned promoter had 18 substitution at 18 sites, 22 deletions at 18 sites and 3 insertion at 3 sites between sites 0---1273 bp which was reported to control temporal-spatial specific expression , and 3 substitution at 3 sites , 6 deletions at 6 sites between -1095 bp and -1273 bp, the key functional sequence area, in comparing with known Osg6B. Based on the sequence analysis, the temporal-spatial speciality of Osg6B’ was then analyzed with transgenic tobacco plants. A binary plant expression vector with Osg6B’ driving report gene GUS was constructed and transferred into tobacco via Agrobacterium tumefaciens mediation. In TO mature transgenic plants tested, GUS staining was observed in different development stages and positions of the anther and some times in calyx, petal, stigma, anther filement and pollens. In 9 TO young plants GUS-tested, 2 plants were negative and the rest were positive: 4 plants stained in root, stem and leaves and 3 plants only in stem and leaves. GUS staining was also found in root, stem and leaves of Kan-resistant Tl seedlings that occurred 3/4 of all Tl generation. These results indicated that the cloned Osg6B’ had a constitutive characteristics but not anther specificity, and the transgene was segregated in a Mendelian fashion in the Tl generation.In the part II, a gene, argE, was cloned from E.coli and sequenced. It had 1152 bp in length, and predictably encoded for Af-acetylornithinase (NAO) with total 383 amino acids. Its DNA sequence and predicated amino acid sequence were entirely identical to those of reported argE. This gene, artificially put under the control of CaMV 35S, was introduced into tobacco with the aid of Agrobacterium, and the transgenic plants obtained were identified by GUS staining, PCR and Southern blot analysis. Its function in transgenic plants is being exploited.更多还原