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题名: 格尔德霉素及美登木素的生物合成研究
作者: 珠娜
学位类别: 博士
答辩日期: 2007-05-30
授予单位: 中国科学院昆明植物研究所
授予地点: 昆明植物研究所
导师: 郝小江
关键词: 安莎类I型聚酮 ; 格尔德霉素 ; 美登木素 ; 内生放线菌 ; 滑桃树
学位专业: 植物学
中文摘要: 本论文由三部分组成,第一部分关于格尔德霉素生物合成途径中后修饰基因的功能鉴定,包括1) 实现在野生型格尔德霉素产生菌中将依赖FAD的单加氧酶基因gel7和氨甲酰基转移酶基因gel8同时敲除获得格尔德霉素前体,验证了gel8编码氨甲酰基转移酶在C-7位引入氨甲酰基;表明格尔德霉素中C-4、C-5之间的不饱和双键是在后修饰反应过程中形成;gel7与后修饰过程中的氧化反应相关。2) 为验证gel7基因功能进行的基因回补及异位表达实验,没有检测到预期产物,说明格尔德霉素前体的氧化过程可能并非一步完成,或者不仅仅是由gel7独立完成。3) 细胞色素P450氧化酶基因gel16的回补实验结果验证了gel16负责格尔德霉素C-4, C-5位的双键形成。4) 利用安丝菌素合成后修过程中N-甲基转移酶基因asm10和卤化酶基因asm12的异位表达实现了对格尔德霉素产生菌突变体产物进行结构修饰。 论文第二部分,为验证“美登木素生物合成的植物内生菌起源”的推想,作者从滑桃树不同组织分离得到内生放线菌约160余株,对其中27个菌株做了16S rDNA鉴定,有一株Br2y-1与“未培养细菌”同源性为96%;针对美登木素活性及结构特征对内生菌发酵产物进行了抗真菌活性、薄层层析及LC-ESI-MS检测,有10株内生菌三种检测均呈阳性;从基因水平以PCR检测根据形态分组的28个代表菌株,有4株具有AHBA合酶基因,其中一株M27m3同时具有氨甲酰基转移酶基因,可能具有合成美登木素类化合物的潜能。对菌株产物进行的抗人体病原菌活性检测结果显示:75%具有抗金黄色葡萄球菌活性,约19%具有抗结核分支杆菌活性,约18%具有抗白色念珠菌活性,并且部分菌株具有2种或2种以上抗菌活性。有10株内生菌对P-388小鼠白血病及A-549人肺癌细胞具有抗肿瘤活性;并且从一个菌株发酵产物中分离得到一个新的吡唑类生物碱。 在第三部分中作者对聚酮类抗生素生物合成研究进展做了文献综述。
英文摘要: The dissertation was composed of three parts. In part A, the study of post-PKS modification gene functions in geldanamycin biosynthesis included: 1) Both gel7 (FAD-dependent monooxygenase gene) and gel8 (carbamoyltransferase gene) were double disrupted in geldanamycin producing strain S. hygroscopicous to genernate progeldanamycin, which provided the evidences that gel8 was working on C-7 carbamoylation, the double bond between C-4 and C-5 was formed during the post modification and gel7 was related to the oxidations in post modification of geldanamycin biosynthesis. 2) In study of gel7 gene function, complementary and ecotopic expression experiments were carried out, however, no expected products could be detected by HPLC or LC-MS, which suggested that there might be more than one oxidations or not only gel7 working in the oxidations on the benzene ring during post modification. 3) Results of gel16 complementary experiment proved that gel16 was working on the formation of double bond between C-4 and C-5. 4) Expression results of asm10 (N-methyltransferase gene) or asm12 (halognase gene) from ansamitocin producing strain in S. hyg M mutant implied the occurrence of the modifications by expression of cloned genes. Part B, the preliminary studies on the hypothesis of endophytic origin of plant maytansinoid focused on searching for the maytansinoid producing microbe(s) among endophytes isolated from Trewia nudiflora. Several endophyte isolation methods were applied to obtain about 160 endophytic actinomycetes isolates from roots, stems, seeds and callous of T. nudiflora. One isolate Br2y-1 out of 27 which were identified by 16S rDNA taxonomy showed 96% homology to uncultured bacteria. Metabolites of 10 isolates showed positive results in screenings with antifungal assay, TLC and LC-ESI-MS detection according to the activity and structure characteristics of maytansinoid. 28 representative isolates from 20 morphological groups were screened by PCR with AHBA (starter unit of maytansinoids) and CT (carbamoyltransferase) gene primers. 4 isolates showed AHBA positive, and among them strain M27m3 also showed CT positive, which suggested it might have the potential to produce maytansinoid. In the bioassay against other human pathogens, 75% isolates showed activity against Staphylococcus auteus, 19% against Mycobaterium tuberculosis and 18% against Candida albicans, besides, some of them showed more than 2 kinds of antimicrobial activities. There were also 10 strains showed antitumor activities against P-388 and A-549 cell lines. A new pyrazole alkaloid was isolated from an endophytic Streptomyces strain. In the last part, the author reviewed the biosynthesis of polyketide.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.kib.ac.cn/handle/151853/62
Appears in Collections:昆明植物所硕博研究生毕业学位论文_学位论文

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Recommended Citation:
格尔德霉素及美登木素的生物合成研究.珠娜[d].中国科学院昆明植物研究所,2007.20-25
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