【作者】 陶美凤；

【导师】 邓子新；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 1999， 博士

【摘要】 链霉菌FR-008聚酮合酶(PKS)基因簇负责七烯大环内酯抗真菌抗生素杀假丝菌素(FR-008)的生物合成，将这个巨大的基因簇转入植物以探索产生抗真菌植物品种的可能性是我们深入研究杀假丝菌素生物合成基因簇的长远目标之一。 为探索在植物中表达极高G+C含量的PKS基因的可能性，构建了携带有编码FR-008 Ⅰ型PKS模块氨基端部分的基因的表达质粒，包括一个酮基合酶(KS)和部分酰基转移酶(AT)活性结构域。该载体携带有花椰菜花叶病毒35S RNA的启动子以指导PKS的表达。由于载体中2.7 kb的PKS基因编码了两种酶活性的结构域而不是完整的Ⅰ型PKS模块，因此我们目前并不期望这个截短的PKS基因在植物能产生抗真菌活性。 为在大肠杆菌超量表达该2.7 kb PKS基因所编码的两个功能结构域，构建了具有不同启动子组合的PKS表达质粒。其中一些可超量表达具有预期分子量的PKS蛋白。进一步截短的基因可以表达预期大小的50 kDa蛋白，表明这些超量表达的蛋白是由克隆在质粒上的PKS基因所编码。 接着将大肠杆菌表达的PKS作为抗原制备与天然FR-008 PKS对应的多克隆抗体。蛋白质印迹实验显示，部分提纯的抗体样品与大肠杆菌中表达的PKS蛋白有特异性反应。同时，这些抗体也可与天然存在于链霉菌FR-008的、巨大的PKS蛋白特异性地反应。这说明构建的大肠杆菌表达质粒中FR-008 PKS基因是以正确的读码框架克隆和表达的；同时也表明抗血清对天然的FR-008 Ⅰ型PKS具有特异性。利用抗体检测PKS的检出极限比考马斯亮蓝染色高1000倍，应可用于检测植物中低水平的PKS表达。 构建了两个链霉菌-大肠杆菌双功能PKS表达质粒，在重组PKS基因上游携带有λ噬菌体启动子。利用这些质粒在变铅青链霉菌和大肠杆菌中均表达出PKS蛋白，两种宿主中的表达都是热依赖的。暗示λ噬菌体启动子和温敏型λ阻遏物在变铅青链霉菌和大肠杆菌中都具有功能。表达PKS的变铅青链霉菌菌株在孢子形成和抗生素产生方面与没有2.7 kb PKS基因的菌株相比较是有区别的，因而推测在链霉菌中表达的双功能结构域PKS蛋白具有活性。 构建了带有λ噬菌体启动子和特异性抗原决定簇(表位)及2.7 kb PKS基因的整合型质粒，用于标记天然的FR-008 PKS。将这些质粒转入链霉菌FR-008的限制性缺陷菌株中得到抗性转化子。Southern杂交证实2.7 kb PKS基因和天然PKS基因之间通过同源重组发生了整合。衍生菌株合成抗真菌抗生素的产量与亲本菌株不同，表明FR-008基因的原始启动子已被改造后的启动子和抗原决定簇所代替。 根据受cIts857调控的P_R能引导PKS基因的成功表达，构建了大肠杆菌表达载体pHZ330以及在大肠杆菌和链霉菌两种宿主中都能表达外源基因的双功能表达载体pHZ1060。两种载体在宿主细胞中是稳定的，应可用于异源基因的表达。更多还原

【Abstract】 A long term goal of our group is to introduce the very large and high G+C content polyketide synthase(PKS) genes for the biosynthesis of the heptaene macrolide antifungal antibiotic Candicidin from Streptomyces species FR-008 into plant to generate fungal resistance lines.An expression vector carrying a fragment encoding the amino-terminal part of an FR-008 Type I PKS module, containing a keto-synthase(KS) and part of an acetyl-transferase(AT) domain was constructed for trial expression of the extremely high G+C content(76 %) PKS gene in plant. This vector containing the promoter of the Cauliflower mosaic virus 35S RNA for the expression of the 2.7 kb PKS gene. Expression of this truncated gene in plant was expected to give information about expression in plant of high G+C content genes but no antifungal activity was expected in this stage.PKS expression plasmids containing strong, temperature-inducible, bacteriophage lambda promoters with or without the Escherichia coli phage T7 promoter were constructed and some of them lead to high expression of the expected c. 100 kDa PKS protein but some of them did not give reproducible high expression in E. coli. Truncating of the 2.7 kb PKS gene from these expression plasmids resulted in production of an expected 50 kDa protein, suggesting that these over expressed proteins were encoded by the cloned PKS gene. The over expressed PKS proteins in E. coli formed inclusion bodies.Then the FR-008 PKS produced in E. coli was used as antigen to raise polyclonal antibodies against the natural FR-008 PKS. Non-specific anti-E. coli antibodies were removed by precipitation with E. coli extract. Western blot experiments indicated that the partially purified antibody preparations reacted specifically with the PKS proteins expressed in E. coli and, as expected, also with the very large PKS proteins naturally present in S. sp. FR-008. This confirmed that the FR-008 PKS gene in the E. coli expression vectors was cloned in the correct reading frame. The above detection of a very large protein in S. sp. FR-008 indicated that the antiserum had the desired specificity for the FR-008 PKS. The detection limitation of PKS using antibodies was 1000 fold higher than Commassie blue staining, ready for detection of the probably very low level of PKS expression in plant.Two bifunctional Streptomyces-E. coli vectors were constructed that contained the phage lambda promoter(PR) upstream of the His6-tagged recombinant PKS gene. Using these vectors, expression of the PKS gene was achieved both in Streptomyces lividans and in E. coli in a heat-dependent manner, suggesting that the lambda promoter and temperature-sensitive lambda represser functioned in S. lividans as well as in E. coli. Also the CI represser was expressed, presumably from its own phage promoter and prevented transcription from PR at low temperature. Moreover, S. lividans strains expressing the c. 100 kDa PKS were different in sporulation and antibiotic production compared to strains without the 2.7 kb PKS gene, suggesting that the PKS protein was active in an unknown manner in Streptomyces.Integrated plasmids containing phage lambda promoter PR -directed and epitope-tagged 2.7 kb PKS gene were constructed for tagging the natural FR-008 PKS with specific immunodeterminant(epitope). These constructs were transferred into Streptomyces sp. DX600, arestriction deficient strain of the antibiotic producer S. sp. FR-008. Integration through homologous recombination between the 2.7 kb PKS gene and the natural PKS gene was confirmed by Southern hybridization. The resulting derivative strains produced antifungal activity in a manner different from the parental strain, indicating the original promoter was replaced with the engineered promoters and the epitopes.Base on the successful expression of PKS gene by CIts857 controlled lambda promoter, an E. coli expression vector pHZ330, an E. coli and Streptomyces bi-functional vector pHZ1060 were constructed containing the dts857-PR for inducible expression in both hosts. Both vector更多还原