【作者】 涂知明；

【导师】 何光源；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 启动子是基因表达调控的重要顺式元件,也是基因工程表达载体的一个重要元件。一些基因只能在特定的组织中表达,就是依赖于组织特异性启动子的功能。无启动子转化策略是一种非常有效的内源启动子的捕获方法,将一个带有UidA基因的无启动子的质粒通过基因枪转化进tritordeum材料中,得到了一批转基因植株。并通过UidA基因的组织化学定位法来判定转基因材料中UidA基因的遗传和表达情况,特别是组织特异性启动子的遗传稳定性。对转基因材料和对照材料的不同组织经X-gluc显色检测UidA基因的表达,包括根、茎、叶和花的各个部分(颖片、外稃、内稃、心皮、花药原基、花粉粒等)的表达情况,成功获得了分别标定有花药原基组织特异性启动子、短细胞特异性启动子、花粉粒特异性启动子和内源广谱启动子的阳性植株。为更明确这些材料的遗传表达特异性,对筛选的这些材料的阳性植株进行了连续的遗传稳定性分析,并进一步用PCR和RT-PCR技术分析UidA基因在不同组织中的分布和发生转录的情况,筛选出了供试材料。用CTAB法提取各种材料叶片的总DNA作模板,在花药原基组织特异性启动子被捕获的材料中,上游使用水稻花药启动子分离的引物P1:5′CACGTAGTTCAATTACAGTTC3′,以UidA基因的部分序列为下游引物P2 :5′ACACAAACGGTGATACGTACACT3′,通过PCR扩增获得UidA基因的上游序列,获得一条长667bp的目的片断,分析发现含有部分UidA基因的序列和一段UidA基因的上游旁侧序列,该序列中具有植物启动子的一些必备元件,包括TATA框,GC框,CAAT框等启动子元件,初步断定它是一段花药组织特异性启动子序列。首次在植物中采用碱性磷酸酶标记DNA制备分子探针。酶在苯醌作用下与单链DNA联结,形成DNA和酶的共价复合物,即酶标探针,此探针通过分子杂交与待测DNA结合,再与酶的底物作用显色,很快便能观察结果。用此探针检测乙肝病人血清中的HBV DNA及转基因植物中的UidA基因,点杂交和Southern杂交结果表明,所合成的酶标探针具有快速、准确、安全而经济的优点。还对三种不同的大肠杆菌(DH5α,TG1和XL1 blue)高效感受态细胞的制备和保藏及转化方法进行了深入的研究。将普通的细菌转化方法做了三种改进:首先是将CaCl2溶液改为TB溶液;其次是将培养基由LB换成S.O.C;再就是在转化过程中加入DMSO或PEG8000。改良的细菌转化方法能大幅提高转化效率。更多还原

【Abstract】 Promoter is a very important element for regulating gene’s expression. It is also a key component of genetic engineering vector. Some genes can only express in a specific tissue, which depends on the tissue specific promoters. The tissue specific promoter’s tagging, testing and isolation methods are studied. Non-promoter strategy to label tissue specific promoter is certified as an effective promoter tagging approach. A non-promoter plasmid with UidA gene was transformed into Tritordeum embryos by bombardment. By analysing the transformed plants, anther primordea, pollen grain, and short cell specific promoters were certified to be labelled by UidA gene.The UidA gene labeled Tritordeum strains have been analysied by using X-gluc to test the expression of UidA gene. By strict detection of GUS activities in different parts of target materials, including root, stem, leaves, flower (glume,Lemma,palea,lodicule,awn,anther primordial, carpel,pollen), some transgenic strains were obtained, and their inheritance also studied in their offsprings. By 5 generation’s genetic analysis and RT-PCR to study the transcription of UidA, several materials labeled with anther primordial, pollen, short cell tissue specific promoters and inner ubiquitious promoter were selected for promoter isolation.A simple PCR method was chosen to isolate the promoters. Using total DNA extracted from leaves of each different strains as template, rice anther specific promoter’primer(P1:5′CACGTAGTTCAATTACAGTTC3′) was used as 5’primer, 3’primer (P2:5′ACACA AACGG TGATACGTACACT3′) is a part of UidA gene,then the sequence including the tissue specific promoter was obtained by PCR reaction. By sequencing and characterizing the target DNA fragment, which contains a part of UidA gene and a flanking sequence, especially some essential promoter elements were found in this sequence. This 667bp DNA fragment containing anther specific promoter was cloned into pGEM-promoter1 vector.A method using alkaline phosphatase to label DNA as probe has been studied and used to detect the HBV DNA in hepatitis patient’s serum and UidA gene in transgenic plants. The modified phosphatase was covalently linked to single stranded DNA using glutraldehyde. Such single stranded DNA enzyme complexes have been tested for blot hybridization and Southern blot, after hybridization and incubation with a substrate solution, result can be visualized directly in one hour. It is a sensitive, specific, rapid, safe and economical probe labeling and detection method.An efficient plasmid preparation and bacterium transformation system has been established. Three different kinds of bacteria (XL1 blue, TG1 and DH5α) have been tested. Each bacterial strain has its best condition depending on its growth curve. The storage time of competent cell and its correlation to transformation rate has been studied. The three crucial alterations to previous methods are the changing of the TB solution to normal CaCl2 solution, the changing of the medium from LB to S.O.C, and addition of DMSO or PEG8000 into transformation system. Improved bacterium transformation system can raise the transformation rate sharply.更多还原