1、紅樹林來源 紅樹林來源 紅樹林來源 紅樹林來源 Streptomyces Streptomyces Streptomyces Streptomyces sp.OUC6819 sp.OUC6819 sp.OUC6819 sp.OUC6819 中多烯大環(huán)內(nèi)酯類抗 中多烯大環(huán)內(nèi)酯類抗 中多烯大環(huán)內(nèi)酯類抗 中多烯大環(huán)內(nèi)酯類抗生素的生物合成研究 生素的生物合成研究 生素的生物合成研究 生素的生物合成研究摘要 摘要 摘要 摘要Streptomyce

2、s sp.OUC6819 分離于我國廣東省紅樹林保護(hù)區(qū)蘆葦根際土壤，能 夠 產(chǎn) 生 一 類 具 有 抗 真 菌 活 性 的 32 元 多 烯 大 環(huán) 內(nèi) 酯 類 化 合 物 (PolyeneMacrolides，PEMs)。本論文以 Streptomyces sp.OUC6819 為研究對(duì)象，主要圍繞PEMs 的生物合成開展了以下研究工作：首先，對(duì) Streptomyces sp.OUC6819 最適的生長條件進(jìn)行了摸索。在不同培養(yǎng)基(

3、MS，ISP2，ISP4，COM，高氏)，鹽濃度(0%~10%)條件下培養(yǎng)，實(shí)驗(yàn)結(jié)果確定最佳產(chǎn)孢培養(yǎng)條件為 ISP2 培養(yǎng)基，28℃，鹽濃 3.3%，pH=7.2。通過抗性敏感 實(shí) 驗(yàn) 確 定 了 Streptomyces sp.OUC6819 對(duì) Thiostrepton 、 Kanamycin 和Erythromycin 的敏感性。其次，成功構(gòu)建了 Streptomyces sp.OUC6819 的基因組文庫。從 Streptomy

4、cessp.OUC6819 中提取基因組 DNA，經(jīng)過 Sau3A I 部分酶切，脫磷，回收 35-45kb左右的 DNA 片段，與經(jīng) XbaI 酶切并脫磷，再用 BamHI 處理的載體 SuperCos1相連接， 連接產(chǎn)物經(jīng)包裝蛋白包裝， 然后侵染宿主細(xì)胞 Escherichia coli Trans 10，構(gòu)建成 Streptomyces sp.OUC6819 的基因組文庫。對(duì)該文庫進(jìn)行了質(zhì)量鑒定，結(jié)果表明：文庫的效價(jià)達(dá) 6.0&#

5、215;106cfu/mL，經(jīng) EcoRI、BamHI 和 XhoI 酶切驗(yàn)證基因文庫隨機(jī)性良好。 所建文庫的各項(xiàng)指標(biāo)均達(dá)到要求， 克隆子分裝保存于－80℃冰箱。 基因組文庫的構(gòu)建為次級(jí)代謝產(chǎn)物生物合成基因簇的克隆， 基因功能研究以及異源表達(dá)奠定了基礎(chǔ)。接著，對(duì) Streptomyces sp.OUC6819 中多烯大環(huán)內(nèi)酯類生物合成基因簇進(jìn)行了克隆與生物信息學(xué)分析。 研究結(jié)果表明， 多烯大環(huán)內(nèi)酯類化合物生物合成整個(gè)基因簇共 98kb，

6、由 14 個(gè)開放閱讀框(Open Reading Frames，ORFs)組成，其中有 4 個(gè) I 型聚酮合酶(Polyketide synthase，PKS)基因(opeG、opeH、opeI 和 opeJ)，5 個(gè)可能的調(diào)控基因(opeA、opeC、opeD、opeE 和 opeF)，1 個(gè)抗性基因(opeK)，1 個(gè) II 型硫酯酶基因(opeM)，1 個(gè) CoA 連接酶基因(opeN)和兩個(gè)未知功能基因(opeB 和 opeL)

7、。本論文采用 PCR targeting 方法構(gòu)建了用于調(diào)控基因 opeA、opeCStudy Study Study Study on on on on the the the the biosynthesis biosynthesis biosynthesis biosynthesis of of of of polyene polyene polyene polyene macrolides macrolides macrolid

8、es macrolides from from from frommangrove mangrove mangrove mangrove- - - -derived derived derived derived Streptomyces Streptomyces Streptomyces Streptomyces sp.OUC6819 sp.OUC6819 sp.OUC6819 sp.OUC6819Abstract Abstract

9、Abstract AbstractStreptomyces sp.OUC6819 was isolated from reeds rhizosphere soil inGuangdong province of China, which is able to produce a series of 32 memberedpolyene macrolides (PEMS) with significant antifungal activ

10、ities. For the purpose ofelucidating the biosynthesis mechanism of polyene macrolides in Streptomycessp.OUC6819 , in this thesis, the following experiments were carried out:Firstly, growth conditions for Streptomyces sp.

11、OUC6819 were studied andoptimized. Streptomyces sp.OUC6819 was inoculated in the different medium (MS,ISP2, ISP4, COM, gause) with salt concentration ranging from 0% to 10%. Theresults of experimental showed the optimum

12、culture conditions are ISP2, 28 ℃, saltconcentration 3.3% and pH=7.2. The antibiotic resistance experiment showedStreptomyces sp.OUC6819 is sensitive to Thiostrepton, Kanamycin andErythromycin.Secondly, the genomic libra

13、ry of Streptomyces sp.OUC6819 was constructed.Total DNA of Streptomyces sp.OUC6819 was extracted and partially digested byrestriction endonuclease Sau3AI, and then DNA fragments at the size of 35-45kbwere recovered and l

14、igated with SuperCos1, which was digested by XbaI,dephosphorylated and redigested by BamHI. The ligation product was packaged intophage particles with Lambda Packaging Extracts, which was then transfected E. coliTrans l0

15、 to yield the genomic library of Streptomyces sp.OUC6819. The titer of thelibrary was 6.0×106 cfu/mL, .meeting the requirements as a standard genomic library.The clones were stored at -80℃. Construction of the genom

16、ic library of Streptomycessp.OUC6819 laid the foundation for cloning of biosynthesis gene cluster of secondarymetabolites and their heterologous expression.Thirdly, the biosynthesis gene cluster encoding polyene macrolid