【作者】 陈双臣；

【导师】 邹志荣； 李君明；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2006， 博士

【摘要】 作为重要的模式经济作物,番茄基因组较小(950MB,编码35000个基因,大部分基因位于占整个基因组25%的常染色质部分),遗传学基础雄厚,并且具有许多拟南芥、水稻等模式植物所不具备的生物学现象(如果实的发育、成熟过程等)。番茄突变体库的建立将为具有重要农艺性状基因的克隆及具有自主知识产权的相关基因的获得奠定基础,所获得的突变体和功能基因组学的研究将为其它蔬菜、水果(尤其是浆果类如Citrus、grape)等重要经济作物的同类研究提供理论和方法上的直接借鉴。本实验旨在通过农杆菌介导的遗传转化,将构建的激活表达标签转化Micro-Tom及M82,获得插入突变群体,并开展对突变体的鉴定和分析,以期分离克隆相关基因。主要研究结果如下:1.分别以CaMV 35S强启动子和Ac转座酶自身携带的弱启动子启动,以GFP、Lc、GUS基因作报告基因,构建了7个激活表达标签载体。2.利用农杆菌介导的遗传转化法将表达载体导入番茄,优化了Micro-Tom的转化体系,获得转基因番茄群体,平均转化效率为40.1%,其中载体pAcGFP转化效率最高,为49.21%。含有GUS基因的表达载体GUSL转化过程中,愈伤不分化,未得到转基因植株。同时利用M82建立了Ac/Ds标签插入辅助群体。3.对转化群体进行了GFP基因活性、染色体倍性及分子检测,结果表明:GFP基因已经整合到番茄基因组中,GFP基因的表达具有组织特异性和遗传材料特异性。气孔保卫细胞叶绿体数表明:转化株染色体为二倍体,倍性没有发生改变。Basta抗性试验表明T-DNA在番茄染色体中平均插入位点数为1.8个;通过Southern杂交分析,26.0%为1个插入位点,51.0%为2个插入位点,T-DNA在番茄基因组中的平均拷贝数为2.2个。对T1代植株的Southern杂交检测表明,Ds因子在基因组中可稳定遗传给子代。Basta抗性检测与Southern杂交检测结果基本一致。Southern杂交和Basta抗性检测还表明,来自同一愈伤组织的转基因植株不一定来自同一克隆。4.利用PCR方法检测杂交群体中Ds因子的转座活性,对280株T2植株的转座情况进行检测,筛选出既含Ds因子又含转座酶Ac基因的植株94株,结果表明,其中有21株Ds因子从原插入位点切离,转座频率为22.3%。利用半巢式PCR对Ds因子的跳跃活性进行了确定。利用TAIL-PCR对转Ds因子的T0植株的T-DNA插入位点的旁侧更多还原

【Abstract】 Tomato has been considered as a model for genomic study of Solanaceae because of its difference to Arabidopsis thaliana and rice in biology phenomena, its smaller genomic size (950 Mb) and powerful genetics groundwork. Construction of insertion mutant pool and function genome research will greatly devote to gene clone with important agriculture character and the related gene with independent knowledge property right. The gained mutants and their function genome research will supply theory and technique references for the study of the other vegetables and fruits, especially for berry fruit like Citrus and grape.The aim of this thesis was to generate a population of T-DNA insertion mutants via Agrobacterium-mediated genetic transformation using the constructed activation tagging into Micro-Tom and M82. With the identity and analysis of mutants, we expected the related genes would be isolate and clone in the coming work. The main results obtained were as followed:1. Seven activation tagging constructs were built promoted with CaMV 35S and Ac self-promoter, and with GFP, Lc, GUS as reporter genes.2. The constructs were transformed into Micro-Tom via Agrobacterium-mediated, and the system was optimized. The transformed tomato population was gained and the mean transformed efficiency was 56.7%. The transformed efficiency of pAcGFP was the highest construct, which reached 49.21%. In the process of GUSL transformation, no any transformed plant was gained because of the callus without differentiation. The assisted Ac/Ds insertional population with M82 was also built.3. GFP gene activation, diploid plants and molecular detection were conducted with the transformed plants. The results showed that GFP gene was interknitted into tomato genome. Expressions of GFP gene possess tissue and genetic material specific. The transformed plants were diploid plants, which was the same as the wildness Micro-Tom. Basta resistance experiment indicated that the average T-DNA inserted sites were 1.8 as the average inserted sites were 2.2 through southern blotting. Analyzed with southern blotting, about 26.0% among T1 population with one inserted site and 51.0% with two inserted sites, the average inserted T-DNA sites were 2.2. It showed the similar results as that by Basta analysis.4. The excision frequency of Ds element in cross-derived population was evaluated with更多还原