【作者】 夏海洋；

【导师】 杨长举；

【作者基本信息】 华中农业大学 ， 动物学， 2006， 博士

【摘要】 放线菌(actinomycetes)属于革兰氏阳性细菌中的高G＋C％类群，具有复杂的发育和分化生长周期，产生大约8700种抗生素和生理活性物质，并拥有独特的遗传特征，是微生物领域基础和应用研究的一个重要材料。本文以重要应用价值的除虫链霉菌(Streptomyces avermitilis)和尤马马杜拉放线菌(Actinomadura yumaensis)、模式链霉菌—天蓝色链霉菌(Streptomyces coelicolor)、有独特染色体外遗传因子的链霉菌44414和诺卡氏菌(Nocardia)107等为材料，开展了功能基因组、抗生素生物合成和DNA复制等方面的研究。具体结果包括：1有序排列的S. avermtitilis基因组文库的构建及多拉菌素(Doramectin)基因工程菌株的获得。以柯斯质粒SuperCos1为载体，构建了除虫链霉菌的基因组文库。对约1000个柯斯质粒上插入片段的两端进行测序，并利用全基因组序列的信息，构建了覆盖约91％基因组的有序排列的柯斯文库。利用λRED高效DNA重组和大肠杆菌-链霉菌接合转移技术，建立了除虫链霉菌的基因组遗传操作系统。可以在该菌中进行高效、精确的基因或基因簇的敲除工作，还能够进行连续的基因缺失研究。运用该系统对几个除虫链霉菌工业生产菌株的次生代谢途径和代谢流向进行了改良，首次获得了遗传改造的可以产生多拉菌素的基因工程菌株。此外，发现aveD和aveF之间可能存在一个前人忽视的基因，发现aveC基因可能直接参与Avermectin的生物合成。2利用抗生素生物合成正调控基因改造Avermectin和Doramectin的工业生产菌。通过将在模式链霉菌中发现的afsR、aveR、orfX、afsB、cprB、metK等调控基因导入到S. avermtitilis的产生Avermectin工业菌株和Doramectin的突变菌株，发现3种调控基因afsR，aveR和orfX对菌株MMR630阿维菌素的产量均可以提高约1倍。但是，以上的3种，加上另外3种调控基因，分别导入菌株G11后，发现除afsB提高约13％外，其余调控基因使菌株多拉菌素的产量反而有不同程度的降低。将调控基因afsB置于链霉菌强启动子permE~*下表达降低了菌株G11中多拉菌素的含量。这些结果表明，调控基因对于不同链霉菌的抗生素生物合成具有不同的影响，反映了抗生素生物合成确实受到了复杂网络的调控。3利用新型载体构建基因组文库以进行DNA大片段的敲除。为了对链霉菌基因组进行DNA大片段的敲除，构建了新型的柯斯载体pHAQ31。利用该载体构建了S. avermitilis和S. coelicolor的有序排列的基因组文库，覆盖率分别为73％和62％。利用这二个文库除了可以进行精确的λRED介导的基因替换敲除外，还可以进行DNA大片段的敲除。在S. avermitilis中，利用该系统不带抗性标记，敲除30kb片段的效率可达10％以上。利用该体系尝试了S. coelicolor多个染色体大片段敲除。利用pHAQ31和SuperCos1构建的两套S. avermtitilis基因组文库，构建了敲除S.avermtitilis的除Avermectin合成以外9个聚酮类抗生素生物合成基因簇载体，并完成了部分8基因簇的敲除工作。4马杜拉霉素(Maduramicin)生物合成基因簇中聚酮合成相关基因片段的克隆。以pHZ1358为载体构建了马杜拉霉素产生菌A. yumaensis的基因组文库。利用从该菌中克隆到的内源探针，对基因组文库筛选获得了9个阳性克隆，通过阳性克隆的两端测序分析，发现4个克隆两侧显示出与聚酮合成酶高度同源，推测可能和马杜拉霉素的生物合成相关。5链霉菌温度敏感性线型质粒pRL4的复制功能研究。链霉菌44414携带3个内源线型质粒，在40度培养条件下，其中pRL4质粒发生丢失(温度敏感性)。通过部分酶切和克隆鉴定了pRL4的基本复制区域，由一个与S. avermtilis中线型质粒SAP1相似的新的基因和一段非编码区组成。通过比较鉴定了S.avermtilis中线型质粒SAP1的中心基本复制区。6诺卡氏菌环型质粒pXT107的复制研究。在Nocardia sp. 107中发现质粒pXT107是诺卡氏菌属中报道的最小的环型质粒之一，由4335bp组成。序列分析及功能研究显示，该质粒具有典型的滚环复制质粒的特征。构建的大肠杆菌—诺卡氏菌穿梭质粒pHAQ22能够在N. corallina 4.1037中复制，但不能在变青链霉菌(S.lividans)中复制。7 A. yumaensis染色体基本复制区的克隆及分析。利用根据dnaA和dnaN保守序列设计的简并引物在马杜拉放线菌中扩增到1.3kb片段。研究发现，尤马马杜拉放线菌染色体复制区位于保守基因dnaA与dnaN之间，包含919碱基对。与已发表的3个属的放线菌染色体oriC序列的特征不同，尤马马杜拉放线菌的染色体oriC含有14个DnaA盒子和2个AT富含区，DnaA盒子的保守序列是(T／C)(T／C)GTCC(A／C)CA。携带该oriC片段的大肠杆菌质粒可以在天蓝色链霉菌中复制和以低拷贝方式遗传。比较来自放线菌4个属的oriC，发现以oriC序列和以16S rDNA序列构建的进化树十分相似，进一步支持了前人的推测。更多还原

【Abstract】 The actinomycetes, filamentous bacteria belong to high G+C content gram-positive group, have a complex morphological development and differentiation life cycle with unique genetics characters. And produce more than 8700 kinds of antibiotics and physiological activity chemical. So actinomycetes were taken as important materials in fundermental and application research on microorganism. In this thesis, investigation on functional genome, antibiotic biosynthesis and DNA replication were carried out on Streptomyces avermililis and Actinomadura yumaensis (with high commercial values), Streptomyces coelicolor (the model actinomycetes), Streptomyces. sp.44414 and Nocardia sp.107. The results are following:1 The construction of an ordered genomic library of S. avermitilis and obtainment the genetic-engineering strain to produce Doramectin. A genomic library of S. avermitilis was constructed with the cosmid vector SuperCosl. After sequenced the. both ends of 1000 cosmid clones, an ordered cosmids library covered more than 91% chromosome of S. avermitilis was obtained on the basis of its genome sequence. Base on the cosmids library, a genetic manipulation system were established through theλRED mediated recombination and Escherichia coli-Streptomyces intergeneric conjugative transfer technique. Efficient and precised gene or gene cluster knockout and sequential gene knockout can be carried on in S. avermitilis with this system. An unprecedented genetic modified strain can produced Doramectin was obtained through secondary metabolism engineering and metabolic flux improvement with this system. In addition, a previous overlooked gene between aveD and aveF genes was discovered, aveC might involve in the biosynthesis of Avermectin.2 Genetic modification of industrial strains of Avermectin-and Doramectin-producers by using positive regulatory genes. Here we reported that several known regulatory genes, afsR, aveR, orfX, afsB, cprB and metK, were cloned into an integrative vector and introduced by conjugal transfer from E. coli to the Streptomyces avermitilis industrial stains that produced Avermectins or Doramectin. The Avermectin Bla yield in strain MMR630 increased about one fold after introduced individually the regulatory genes aveR, afsR or orfX. But strain G11 with the introduced individual 6 genes, including the former three genes, decreased the yield of Doramectin except the afsB gene increased 13%. Expression of afsB gene under the constitutive promoter permE~* decreased the productivity of Doramectin in strain G11. These results indicate that the regulatory genes have differential effects on the antibiotic production of different industrial strains, and suggest that regulatory of antibiotic biosynthesis indeed involves in a complicated network in Streptomyces.3. Establishment large chromosome fragment deletion on the basis of genomic library constructed with novel cosmid vector. Cosmid vector pHAQ31 was constructed to carry out large DNA fragment deletion research. Ordered cosmids library of S. coelicolor and S. avermtilis constructed with the conjugative cosmids vector pHAQ31 covered each genome 73% and 62% respectively. Both libraries can carry out large fragment deletion besides theλRED mediated gene PCR-targeting system. For example, 30kb fragment no-marker deletion efficiency is more than 10% with this system in S. avermitilis. Large chromosome fragment longer than 80kb deletion were also test in S. coelicolor. Nine vectors were constructed to delete 9 ploketide biosynthetic gene clusters except that of Avermectin used both librarys of S. avermitilis constructed with SuperCos1 and pHAQ31. Now complete the deletion of seven PKS clusters.4 Cloned DNA fragments related with the biosynthetic gene cluster of Maduramicin. A cosmids genomic library of A. yumaensis NRRL12515 constructed with cosmid vector pHZ1358. Nine positive clones were obtained through screened the library by in situ colony hybridization with the endogenious (3-keto-synthase gene fragment probe,which was amplified with designed degenerate primers on conserved motif in this strain. After both ends seuqnecing, the ends of four clone showed high homologous with PKS genes. It canbe concluded that these 4 clones probably related with the biosynthesis of Maduramicin.5 Replication of a temperature-sensitive linear plasmid pRL4. Streptomyces sp. 44414 harbored three linear plasmids, and one of them was lost during culturing at 40℃(temperature-sensitive). By partially digested plasmid DNA, cloned into an E. coli plasmid pQC156 and introduced by transformation into Streptomyces lividans ZX7, a replication origin of pRL4, consisting of a linear plasmid SAP1-like gene and a non-coding sequence, was identified. As expected, the SAP1 gene and adjacent non-coding sequence also contained activity of replication.6 Studies on the Nocardia circular plasmid pXT107. We report here a new identified plasmid pXT107 of Nocardia sp.107, one of the smallest circular plasmid found in Nocardia. The complete nucleotide sequence of pXT107 consisted of 4335-bp with 65% G+C content, encoding one Rep (replication protein) and six proteins of unknown functions. The Rep, double strand origin (dso) and single strand origin (sso) of pXT107 resembled these of typical rolling-circle-replication plasmids, such as pNI100 of Nocardia, pRE8424 of Rhodococcus and pIJ101 of Streptomyces. An E. coli-Nocardia shutter plasmid pHAQ22, containing the rep gene, propagated in Nocardia but not in Streptomyces.7 Cloning and functional studies of the chromosomal replication origin(oriC) of Actinomadura yumaensis. A 1.3kb fragment amplified in A. yumaensis with degenerate primers designed on the conserved motifs of dnaA and dnaN. Result showed that the oriC was located between the conserved genes dnaA and dnaN of chromosome and consisted of 919 bp. Unlike previous published actinomycetes oriC, the oriC of Actinomadura yumaensis contained 14 DnaA-boxes with conserved 9-bp sequence (T/C)(T/C)GTCC(A/C)CA and 2 AT-rich sequences. An E. coli plasmid carrying the oriC fragment was able to propagate in Streptomyces coelicolor in a low copy number. Phylogenetic trees based on the oriC sequences of four genera actinomycetes and of 16S rDNA were similar, supporting the previous hypothesis.更多还原