【作者】 胡敏珊；

【导师】 张义正；

【作者基本信息】 四川大学 ， 遗传学， 2001， 硕士

【摘要】 首先测定了粗毛栓菌(Trametes gallic)对潮霉素的敏感性。结果表明，100μg／mL的潮霉素便足以完全抑制粗毛栓菌在三种常用的具有代表性的培养基上的生长。 将粗毛栓菌总DNA经Sau3Al酶切后插入到启到子探针型载体pSUPV8的BamHI位点，转化大肠杆菌(Escherichia coli)XL-1 Blue,获得具有潮霉素抗性的重组子8个，依次命名为pTP1-pTP8。限制酶切分析表明它们均含有插入片段，大小为0.6-1.7kb。各基因启动子在大肠杆菌细胞中的起动效率采用潮霉素抗性水平进行测定，最高者可达350μg／mL，最低者为150μg／mL。对重组质粒pTP6进行了限制酶谱分析，结果表明，TP6片段长1.7kb，内有KpnⅠ、XbaⅠ和SacⅠ位点各一个。对TP6片段进行的次克隆分析表明，去掉其5’-端和0.8kb和1.2kb片段后，其潮霉素抗性由250μg／mL均降为150μg／mL。推测该0.8kb片段中可能存在一个正调控序列。用TP5片段作为探针与BamHI和EcoRI酶切的粗毛栓菌总DNA进行Southern杂交结果表明，TP5片段来源于粗毛栓菌总DNA。对TP5的3’-端进行的序列分析结果表明，在600bp的序列中，存在典型的真核生物基因启动子结构特点，同时也具有与原核生物基因启动子相似的一些保守序列。实验结果还表明，潮霉素磷酸转移酶基因5’-端编码区核苷酸序列的改变，并不影响基因表达产物的生物学活性，证明该基因可用作报告基因用于基因启动子的分离，启动子效率的测定以及其它DNA调控片段功能的研究等。更多还原

【Abstract】 First of all, the sensitivity of Trametes gallic to hygromycin B was determined, the result showed that a hygroroycin B concentration of 100μg/mL can completely inhibit the growth of T. gallic at three representative culture media.Promoter-probe vector pSUPV8 was used to clone promoters from T. gallic. The chromosomal DNA of T. gallic digested by Sau3AI was ligated with BamHI-cut pSUPV8. The ligated mixture was used to transform E. coli XL-1 Blue. Eight hygromycin B -resistant clones (named pTP1-pTP8) were obtained. Restriction analysis indicated that all eight recombinants had inserts whose sizes ranged from 0. 6 to 1. 7kb. Their hygromycin B -resistant level was determined on different concentrations of hygromycin B-containing plates. The results demonstrated that the hygromycin B resistance level of these recombinants ranges from 1,50 to 350μg/mL. The recombinant pTP6 was chosed to be analysed via 10 different kinds of restriction enzymes. The result indicated that there are three restrition sites, KpnI, XhaI and Sad in the TP6 fragment with 1.7kb in size. Subcloning analysis of TP6 showed that deletion of the 0. 8kb or the 1. 2kb of 5’-terminal segment of pTP6, decreases the hygromycin B~resistant level from 2.50 to 1.50μg/mL It is deduced that there may be an upstream activator sequence in this 0.8kb segment. Southern hybridization using TP5 as probe showed that TP5 can hybrid with the total DNA of T. gallic but not with that of ?coli, indicating that TP5 fragment is from fromT. gallic. The 3’-terminal segment of TP5 fragment was a] so sequenced, The result indicates that it contains several sequences similar to both prokaryotic and eukaryotic gene promoter sequences. It also showed that the 5’ -terminal segment of hygromycin phosphoric acid transferase can be changed without any influence on the activity of the expressed gene product. It is evident that this gene can be used as a reporter gene for the isolation of the promoters, determineation of the promoter efficiency and the study of the function of other DNA modulator element.更多还原