【作者】 刘朝莹；

【导师】 吴春笃；

【作者基本信息】 江苏大学 ， 环境工程， 2012， 博士

【摘要】 蓝藻(蓝细菌)是一类古老的具有放氧光合作用的革兰氏阴性细菌,在漫长的自然选择过程中进化出独特的环境适应能力,广泛分布于不同的生境中。由于水体的富营养化,蓝藻常常爆发形成水华,甚至释放毒素,成为世界性的环境问题,引起公众的关注。对磷极高的生物利用率是蓝藻获得生态竞争优势、导致蓝藻水华发生的关键因素。虽然富营养化水体中磷源非常丰富,但蓝藻等微生物能直接利用的无机磷却很缺乏。因此,阐明蓝藻细胞利用不同形式磷源的代谢调控,对揭示蓝藻水华发生的分子机制、监测蓝藻水华发生具有重要的意义。鱼腥藻(Anabaena sp.)FACHB 709是来源于淡水湖泊(中国,武汉东湖)的一种丝状固氮蓝藻。该藻与模式生物鱼腥藻(Anabaena sp.)PCC 7120高度同源,具有遗传操作系统,可通过接合转移将重组基因导入细胞。本论文利用微生物学、生物信息学、生物化学和分子生物学技术,研究了不同磷源对鱼腥藻FACHB 709生长的影响、发现并鉴定了在藻细胞的磷代谢调控过程中具有关键作用的碱性磷酸酶基因,并通过分析多个碱性磷酸酶基因对不同磷源的表达调控及其生理功能,以期揭示鱼腥藻FACHB 709碱性磷酸酶基因在磷代谢调控中的作用机制。本论文包括以下主要研究内容和结果：(1)研究了不同种类、不同浓度磷源对鱼腥藻FACHB 709生长和碱性磷酸酶活性的影响,结果表明磷缺乏诱导藻细胞生长抑制而不是死亡；鱼腥藻细胞通过上调碱性磷酸酶活性应对磷饥饿。磷饥饿细胞在无机磷不存在的条件下,能利用有机磷G-6-P和pNPP获得部分生长。同时,有机磷的利用可在一定程度上导致细胞总碱性磷酸酶活性下降。(2)分析鱼腥藻FACHB 709和其它几种蓝藻的亲缘关系,发现鱼腥藻FACHB709是鱼腥藻PCC 7120和产水华蓝藻—多变鱼腥藻(Anabaena variabilis)ATCC29413的近源种。利用近缘种中公认的碱性磷酸酶基因序列,扩增并克隆了鱼腥藻FACHB 709中可能的碱性磷酸酶基因,对克隆的全基因DNA片段测序和分析,发现鱼腥藻FACHB 709中存在四个碱性磷酸酶基因,分别为phoA-709、phoDl-709、phoD2-709和phoS-709.(3)构建了鱼腥藻FACHB 709碱性磷酸酶基因启动子和β-半乳糖苷酶基因(lacZ)的转录融合重组质粒,并将重组质粒接合转移到鱼腥藻FACHB 709,通过测定所得鱼腥藻调控报告菌株的β-半乳糖苷酶活性,分析四个碱性磷酸酶基因在不同浓度无机磷存在条件下的表达调控。结果表明,无机磷充足条件下,除了phoD2-709没有转录活性外,其余三个碱性磷酸酶编码基因phoA-709、phoD1-709和phoS-709都有转录活性；无机磷缺乏条件下,四个碱性磷酸酶基因通过上调转录水平,参与鱼腥藻FACHB 709的磷代谢调控反应,其中以phoA-709的作用最为显著。(4)利用免疫学方法,研究了鱼腥藻FACHB 709中四个碱性磷酸酶基因在蛋白水平的表达和分布。结果显示无机磷缺乏条件下,四个碱性磷酸酶基因的表达水平全部升高,且它们的表达产物分泌到了细胞外培养基中。(5)构建碱性磷酸酶基因缺失突变株DRTH65 (敲除phoA-709)和DRTH61(敲除phoS-709),检查突变株的生长和生理表型,结果表明在实验条件下,突变株DRTH65和DRTH61的生长和碱性磷酸酶活性与野生型鱼腥藻的没有差异。通过分析单个碱性磷酸酶基因缺失后,其余APase基因的表达调控,发现在含磷和缺磷条件下,phoD1-709和phoS-709转录水平的上调一定程度上可弥补phoA-709的失活；而phoD1-709和phoA-709转录水平的上调则一定程度上弥补了phoS-709的失活。本论文得到以下主要结论：(1)无机磷是鱼腥藻FACHB 709细胞吸收和利用的主要磷源形式和关键生长因子；藻细胞通过调控碱性磷酸酶活性应对无机磷饥饿诱导的细胞生长抑制。(2)鱼腥藻FACHB 709是鱼腥藻PCC 7120的近缘种,并且具有与之相同的碱性磷酸酶基因组成和序列高度同源的碱性磷酸酶PhoA-709、PhoD1-709、PhoD2-709和PhoS-709.(3)磷饥饿诱导鱼腥藻FACHB 709中四个碱性磷酸酶基因phoA-709、phoD1-709、phoD2-709和phoS-709转录水平和翻译水平的上调,但转录水平上调的程度存在差异。其中,phoA-709在藻细胞磷源代谢调控中具有关键作用。(4)鱼腥藻FACHB 709中四个碱性磷酸酶PhoA-709、PhoD1-709、PhoD2-709和PhoS-709都是分泌型蛋白,缺磷诱导后分泌到细胞外培养基中。(5)phoA-709和phoS-709在鱼腥藻FACHB 709细胞磷代谢中具有重要作用但并非必需基因；phoA-709、phoD1-709和phoS-709编码的碱性磷酸酶可能在功能上重叠或互补。更多还原

【Abstract】 Cyanobacteria are an ancient and diverse group of microorganisms found in many different ecosystems. In a long history of adaptation to the extreme or variable environments, cyanobacteria have developed more complicated regulatory mechanisms than those of other prokaryotes. In recent years, due to the eutrophication of water bodies, cyanobacteria often form blooms, even produce toxin. It has been a worldwide environmental problem that causes public attention. The extremely strong bioavailability of cyanobacterial cells for inorganic phosphorus plays a key role in the ecological success of cyanobacteria which causes occurance of cyanobacterial blooms. Phosphorus sources are extremely rich in eutrophic waters, but inorganic phosphorus, which can be directly used by cyanobacterial and other microorganism cells is limited. Studies on the phosphorus metabolic regulation in cyanobacteria are very important to reveal the mechanism for and to monitor the outbreak of algal blooms.An additional filamentous nitrogen-fixing cyanobacterium, Anabeana sp. FACHB 709 (hereinafter FACHB 709), was isolated from a freshwater lake in Wuhan, China (East lake). It is closely related to Anabeana sp. PCC 7120 and able to be genetically manipulated by using conjugal transfer system. In this research, characters of growth and alkaline phosphatases activity of FACHB 709 were investigated, and then the response of the FACHB 709 alkaline phosphatases to inorganic phosphorus starvation was discovered. So, genes encoding alkaline phosphatase were identified in FACHB 709. In order to elucidate their action mechanism in phosphorus metabolic regulation, the expression, regulation and physiological function of the identified alkaline phosphatase genes in FACHB 709 were analyzed under the conditions of different phosphorus sources.This research mainly obtained the following results:(1) The growth characteristics and response of the FACHB 709 to different phosphorus were studied. The results show that inorganic phosphorus starvation induces growth cessation but not cell death. FACHB 709 increase alkaline phosphatase activity in response to the inorganic phosphorus-limiting stress. Phosphorus-starved cells can utilize G-6-P and pNPP to support some growth in the absence of inorganic phosphorus. At the same time, the absorption of organic phosphorus will partially suppress the alkaline phosphatase activity of starved cultures.(2) The analysis of phylogenetic tree based on the 16S rDNA sequences of FACHB 709 and several other cyanobacteria prompts that FACHB 709 is very closed to Anabeana sp. PCC 7120 and the blooms-forming alga Anabaena variabilis ATCC 29413. The DNA sequences of putative alkaline phosphatase genes in FACHB 709 were amplified and cloned, then sequenced and analyzed. The results indicate that there are four alkaline phosphatase genes, phoA-709, phoD 1-709, phoD2-709 and phoS-709 exist in FACHB 709 cells.(3) The recombinant plamids with lacZ transcriptional fusions were constructed and introduced into FACHB 709 via conjugal transfer, producing strains FACHB 709 (pJS795), FACHB 709 (pTH70), FACHB 709 (pTH67), FACHB 709 (pTH68) and FACHB 709 (pTH64), respectively. By determining P-galactosidase enzyme activity of these strains, transcriptional regulation of four alkaline phosphatase genes was analyaed in the presence of different concentrations of inorganic or organic phosphorus conditions. The results indicate that under inorganic phosphorus-sufficient conditions, except phoD2-709, three other alkaline phosphatase genes are functionally expressed, but all four alkaline phosphatase genes, phoA-709, phoDl-709, phoD2-709 and phoS-709 were upregulated transcription to be involved in phosphorus metabolic regulation during phosphate starving, and the performance of phoA-709 was the best.(4) Using the immunological methods, the expression at the protein level and location of four alkaline phosphatase genes from FACHB 709 were studied. The results show that the four alkaline phosphatase genes were upregulated and their products were secreted to the extracellular medium in the absence of inorganic phosphorus.(5) The phoA-709 and phoS-709-inactivated mutants, DRTH65 and DRTH61of FACHB 709 were constructed, and the investigations into the growth and physiological phenotype of mutants were focused on. The results indicate that under inorganic phosphorus-sufficient and -limiting conditions, no difference was observed in the growth rate and the alkaline phosphatase activity between the wild-type strain and the mutant DRTH65 or DRTH61. So, the lacZ transcription fusion plasmids pTH67, pTH68 and pTH64 were introduced into the mutant strain DRTH65 to obtain the strains DRTH65 (pTH67), DRTH65 (pTH68) and DRTH65 (pTH64), respectively. And similarly, the lacZ transcription fusion plasmids pTH67, pTH70 and pTH64 were introduced into the mutant strain DRTH61 to obtain the strains DRTH61 (pTH67), DRTH61 (pTH70) and DRTH61 (pTH64), respectively. By detecting theβ-galactosidase enzyme activity of these strains, the transcriptional regulation of other three active alkaline phosphatase genes was studied. The results show that to some extent, the increased expression of phoD 1-709 and phoS-709 could counteract the phoA-709 inactivation in DRTH65 under experimental conditions, and similarly, expression of phoD1-709 and phoA-709 to some extent could counteract the phoS-709 inactivation in DRTH61 under experimental conditions.On the basis of above results, we conclude as follows:(1) Inorganic phosphorus is the major form of phosphorus source and a key growth factor for the Anabaena sp. FACHB 709 cells. Cyanobacterial cells increase alkaline phosphatase activity in response to the growth inhibition of phosphate starvation-induced cells.(2) Anabaena sp. FACHB 709 is closely related to Anabaena sp. PCC 7120. And this strain also has the same composition and highly homologous sequences of the alkaline phosphatase genes, phoA-709, phoDl-709, phoD2-709 and phoS-709 with Anabaena sp. PCC 7120.(3) Inorganic phosphorus starvation induced the upregulation of four alkaline phosphatase genes, phoA-709,phoDl-709, phoD2-709 and phoS-709 from Anabaena sp. FACHB 709 at the transcription and translation levels, but the increases of their transcription levels were different from one another. phoA-709 plays a crucial role in the phosphorus metabolic regulation of the FACHB 709 cells.(4) The alkaline phosphatase of Anabaena sp. FACHB 709, PhoA-709、PhoD1-709、PhoD2-709 and PhoS-709 all are secreted proteins. They are induced by inorganic phosphorus starvation and released out of the cells into the medium. (5) The mutational analysis showed that phoA-709 and phoS-709 play an important role in phosphate metabolism of cyanobacteria cells but not essential genes, and the function of alkaline phosphatases encoded by the genes, phoA-709, phoDl-709 and phoS-709 probably overlap or complement each other.更多还原