海洋聚磷菌Halomonas YSR-3的除磷特性研究

IOCAS-IR > 海洋环流与波动重点实验室

	海洋聚磷菌Halomonas YSR-3的除磷特性研究
其他题名	Phosphate-Absorbing Characterization of a Marine Polyphosphate-Accumulating Bacterium, Halomonas YSR-3
	任世英
学位类型	博士
	2008-05-25
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	聚磷菌多磷酸盐颗粒盐单胞菌属
摘要	本论文报道从海洋中分离到的一株聚磷菌的分离、鉴定、在系统发育中的地位、除磷特性、菌体内多磷酸盐颗粒的研究、D-海因酶和核苷二磷酸激酶基因的克隆及序列分析，为海水系统的生物除磷提供部分基础资料。从黄海海域分离到聚磷菌Halomonas sp. YSR-3，菌体呈杆状，大小为3.5 μm×1 μm，革兰氏阴性，好氧生长，能运动。透射电镜观察发现，菌体内有致密颗粒。经DAPI染色确定该致密颗粒是多磷酸盐，亦可称为异染粒、迂回体。16S rDNA鉴定结果表明，YSR-3与Halomonas属中的marine bacterium B5-7有较高的同源性，相似值99%。YSR-3的生理生化特性：对氯霉素和卡那霉素敏感；淀粉水解呈阳性；反硝化和几丁质降解呈阴性；能将葡萄糖作为唯一碳源和能源。对YSR-3的培养条件进行优化。以海水2216培养基、24 ℃、180 rpm、pH 6.5的条件培养，更利于菌体生长和菌体内多磷酸盐的形成。对YSR-3的除磷特性进行研究。无磷培养时，菌体不能生长；用磷酸钾盐作为磷源时，菌体生长较好，形成多磷酸盐的菌体比例较高；较适合YSR-3菌体生长和多磷酸盐形成的磷源是KH2PO4，较适磷浓度为1.5 mmol/L。pH的变化影响菌株的生长、多磷酸盐形成和除磷效果。pH值为5时，菌体的数量几乎不增加，体内多磷酸盐和培养基中磷含量变化不大；pH值为6、7和8时，菌体生长良好，95%以上的菌体内形成多磷酸盐，培养基中磷含量明显下降。YSR-3在不同培养基中除磷量和除磷率不同。在高磷培养基中除磷量为0.7 mmol/L(磷含量由1.84 mmol/L降到1.14 mmol/L)，除磷率为37.5%；在低磷培养基中除磷量为0.02 mmol/L(磷含量由0.028 mmol/L降到0.008 mmol/L)，除磷率为72.2%。以海洋聚磷菌Halomonas sp. YSR-3的总DNA为模板，用PCR法扩增D-海因酶基因和核苷二磷酸激酶基因，将扩增片段克隆到pGM-T载体，转化E.coli TOP10菌株，经蓝白斑筛选、菌落PCR得到阳性克隆，测序后对序列进行Blast比对分析。得到的D-海因酶基因序列长度为1510 bp，与Pseudomonas entomophila L48的海因酶基因序列的相似性为77%。翻译后的序列与Pseudomonas fluorescens Pf-5，Marinomonas sp. MED121，Burkholderia vietnamiensis G4的海因酶蛋白序列相似性分别为75%，73%，70%。得到的核苷二磷酸激酶基因序列长度为420bp，翻译后的序列与Loktanella vestfoldensis SKA53，Jannaschia sp. CCS1，Roseobacter sp. CCS2的核苷二磷酸激酶蛋白序列相似性分别为89%，86%，85%。聚磷菌能将外界环境中的磷吸收到体内，并以多磷酸盐的形式储存。多磷酸盐对于细胞的生存和生长有很重要的作用，但目前对于多磷酸盐的形成过程以及过程调控还不是很清楚。在今后可以通过构建高效表达的重组菌，提高与除磷相关的酶的纯度及活性。同时可以将相关酶的基因进行突变，对基因表达的调控以及酶的代谢以及功能结构等多方面进行基础研究，使聚磷菌在生物除磷中得到广泛应用。
其他摘要	Halomonas sp. YSR-3 not only belongs to genus Halomonas, but also can accumulate polyphosphate (polyP) in its cells. This trait is not reported in other strains of the genus Halomonas and this special trait may be important in the research of the phosphorus circulation in the marine system. The ability of strain YSR-3 phosphate-absorbing may be applied in the process of the biological phosphorus removal of seawater. We also researched on the optimum conditions for forming intracellular granules, the phosphate-absorbing characterization, cloning and analyze of D-hydantoinase gene sequence and nucleoside diphosphate kinase (NDPK) gene sequence as the template of strain YSR-3 total DNA. A polyphosphate-accumulating bacterium YSR-3 was isolated from the Yellow Sea. The cell is motile and rod-shape, 3.5 μm×1 μm, Gram negative, aerobic. Phosphate in culture media is absorbed and stored as the form of polyP polymer in cells. On the basis of 16S rDNA analyze, strain YSR-3 belongs to genus Halomonas of γ- Proteobacteria. The cells are susceptible to antibiotics Kn and Cl. The reaction of amylase is positive, but denitrification is negative. The cells can use glucose as sole carbon source and energy source but can not degrade chitin. The cells can be cultivated well as following conditions: 3% (w/v) NaCl, 24 ℃, 180 rpm, and initial pH 6.5. Under various culture conditions the growth and phosphorus absorption of the marine bacterium Halomonas YSR-3 was studied. In phosphorus-free medium, the ΔOD480 of the cells was almost invariable and cells didn’t store polyP granules. YSR-3 grew better and had higher ratio of accumulating-polyP cells in medium containing sodium phosphate than potassium phosphate. When the concentration of KH2PO4 was 1 mmol/L, YSR-3 grew well and absorbed more phosphate with more intracellular polyP granules. When the medium pH value was 5, the stored intracellular polyP granules had not been metabolized. However, when the value was 6, 7 or 8, YSR-3 grew well and beyond 95% cells accumulated polyP, with decreasing phosphorus concentration in supernatant. When YSR-3 was incubated in high-phosphorus medium, the phosphorus concentration in supernatant decreased from 1.84 mmol/L to 1.14 mmol/L, absorbing 0.7 mmol/L phosphorus with the 37.5% phosphorus-absorbing ratio; when YSR-3 was incubated in low-phosphorus medium, the phosphorus concentration in supernatant decreased from 0.028 mmol/L to 0.008 mmol/L, absorbing 0.02 mmol/L phosphorus with the 72.2% phosphorus-absorbing ratio. D-hydantoinase gene sequence and NDPK gene sequence were amplified respectively by PCR technique as the template of YSR-3 total DNA and then inserted into pGM-T vector. The recombinant plasmids were transformed into E. coli TOP10 strain. The positive transformants with D-hydantoinase gene sequence or NDPK gene sequence were obtained by blue-white selection and clony PCR method, and then the positive ones were sequenced. The sequenced fragments of D-hydantoinase gene including 1510 bp and NDPK gene including 420 bp were analyzed by NCBI database. The similarity of D-hydantoinase gene sequences between YSR-3 and Pseudomonas entomophila L4 is 77%. The similarity of YSR-3 translated D-hydantoinase gene sequence to the sequences of Pseudomonas fluorescens Pf-5, Marinomonas sp. MED121, Burkholderia vietnamiensis G4 is 75%, 73%, 70% respectively. The similarity of YSR-3 translated NDPK gene sequence to the sequences of Loktanella vestfoldensis SKA53, Jannaschia sp. CCS1, Roseobacter sp. CCS2 is 89%, 86%, 85% respectively.
页数	103
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/603
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	任世英. 海洋聚磷菌Halomonas YSR-3的除磷特性研究[D]. 海洋研究所. 中国科学院海洋研究所,2008.

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