【作者】 毕相东；

【导师】 包振民； 唐学玺； 邢克智； 周文礼；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2013， 博士

【摘要】 海洋微藻与异养细菌长期共存于海洋生态系统中，具有错综复杂的关系，既存在互利共生，又有相互拮抗。细菌生长过程中自身不断产生且分泌群体感应信号分子，并通过感知群体感应信号分子的浓度来检测周围细菌的数量，当环境中的信号分子的浓度达到一定阈值时，就会启动某些相关基因的表达，促使细菌完成一种群体行为来有效地适应环境压力、协调与宿主的关系等。本研究以小球藻为研究材料，采用实验生态学方法系统地研究小球藻与优势共栖异养细菌间的相互作用及对优势共栖异养细菌群体感应信号分子的响应。研究结论将进一步明确海洋微藻与共栖异养细菌的相互作用规律，进而为揭示赤潮的爆发机理及解决经济微藻的种质退化等实际问题提供科学的方法，最终为恢复海洋生态系统的合理结构及改善养殖池塘的生态环境提供科学的理论依据。研究结果如下：1采用稀释平板涂布法自菌藻共培养体系中分离纯化出7株小球藻共栖异养细菌，并将其分别命名为Z-TG01、Z-TG02、Z-TG03、Z-TG04、Z-TG05、Z-TG06和Z-TG07，其中Z-TG01、Z-TG02和Z-TG03为优势菌株，优势度分别为48.8%、14.0%和18.6%；随后经梅里埃VITEK2COMPACT全自动细菌鉴定仪鉴定表明3株优势共栖细菌Z-TG01、 Z-TG02及Z-TG03分别为少动鞘氨醇单胞菌(Sphingomonas paucimobilis)、泛菌属(Pantoea spp.)及香味菌属（Myroides spp.）；并以根癌农杆菌Agrobacterium tumefaciens KYC55为报告菌株，采用β-半乳糖苷酶法检测表明这3株优势共栖细菌均具有AHLs信号分子活性，且活性随细菌密度增加而升高。2以根癌农杆菌A. tumefaciens KYC55为报告菌株，采用β-半乳糖苷酶法检测表明整个指数期小球藻滤液在2h内均可显著或极显著抑制Z-TG01及Z-TG03菌株AHLs信号分子的活性(p<0.05或p<0.01)，而指数中期小球藻滤液在2h内可显著或极显著抑制Z-TG02菌株AHLs信号分子的活性(p<0.05或p<0.01)，原因可能是本研究所用的小球藻藻株可分泌AHLs信号分子的类似物或淬灭酶，抑制共栖细菌AHLs信号分子的活性，进而干扰群体感应介导的细菌毒素的释放，使小球藻本身处于适宜生长的环境。3在研究小球藻与优势共栖异养细菌间的相互作用规律及藻菌互作过程中优势共栖异养细菌群体感应信号分子活性的变化规律时发现：①当共培养体系中Z-TG01菌株处于一定密度时，小球藻与Z-TG01菌株相互促进生长，指数期的小球藻能够明显抑制Z-TG01菌株AHLs信号分的活性；②不同初始密度的Z-TG02菌株对小球藻的生长均具有一定的抑制作用，且与小球藻共培养期间Z-TG02菌株AHLs信号分子活性未受到明显抑制原因是小球藻的生长受到Z-TG02菌株抑制而使其分泌的AHLs信号分子的类似物或淬灭酶总量较少或小球藻分泌AHLs信号分子的类似物或淬灭酶在共培养体系中缺乏稳定性所致；③当共培养体系中Z-TG03菌株处于一定密度时，小球藻与Z-TG03菌株相互促进生长，指数期的小球藻能够明显抑制Z-TG03菌株AHLs信号分子的活性。4在研究群体感应信号分子对小球藻生长及抗氧化酶系统的影响时发现：①低浓度C6-HSL(50、100及200nmol/L)8h时即可显著提高小球藻的相对生长率(p<0.05)，高浓度C6-HSL(≥400nmol/L)24h时即可显著降低小球藻的相对生长率(p<0.05)，且表现出较强的浓度依赖性抑制作用，表明C6-HSL对小球藻生长的影响表现为低浓度促进而高浓度抑制的效应；②50nmol/L C6-HSL在4h时即可显著提高SOD、POD、CAT及GPX的活性(p<0.05)，同时显著降低MDA的含量(p<0.05)；与低浓度组相比，400nmol/L C6-HSL作用下，藻细胞抗氧化酶活性均经历先升高后降低的过程，24h时藻细胞MDA含量显著升高(p<0.05)。表明低浓度C6-HSL可以激活小球藻细胞的防御性反应，而高于400nmol/L的C6-HSL对小球藻抗氧化系统有一定的损伤作用。5采用iTRAQ技术从蛋白表达水平上研究小球藻对AHLs信号分子的应答机制。结果表明从400nmol/L的C6-HSL处理组(24h)与对照组小球藻中总共鉴定出281个蛋白质，发现差异表达的蛋白质为10个(差异倍数1.2倍以上，5个上调，5个下调)。小球藻光系统II的D1蛋白及热激蛋白90表达量显著下调，表明藻细胞的光合作用等生理功能已经受到不同程度的损伤；与此同时烯醇酶、景天庚酮糖-1,7-二磷酸酶、ATP合酶β亚基及液泡膜ATP酶A亚基的表达都明显上调(p <0.05)，提示在C6-HSL损伤藻细胞光系统的同时，藻细胞加强厌氧性呼吸及ATP合成效率为修复受损的光系统、有毒物质的跨膜转运及藻细胞正常的生长提供能量。另外，硫酯生物合成酶的表达亦显著上调，说明藻细胞正积极修复C6-HSL胁迫下受损的细胞膜。更多还原

【Abstract】 Marine microalgae and numerous heterotrophic bacteria in its phycosphereconstitute a specific microcosm and have complicated ecological interactions witheach other, either mutualism or mutual antagonism. Bacteria can synthesize andrelease quorum sensing signal molecules into surrounding environment, and detect thecell density by sensing the concentration of quorum sensing signal molecules.Bacteria can control gene expression collectively and synchronize group behaviour toadapt to stress the changing environment and coordinate the relationship with the hostwhen the signal molecule concentration reaching to a threshold value.In present study, a common diet microalgae-Chlorella vulgaris was selected toserve as experimental material, and the mutual ecological effects of C. vulgaris anddominant associated heterotrophic bacteria and the response of C. vulgaris to quorumsensing signal molecules were studied under controlled laboratory conditions. Theresults could further clearly defined the mutual action rule of marine microalgae andheterotrophic bacteria, and then supply scientific method to reveal the mechanism ofred tide and solve the problem of genetic degradation of marine economic microalgae,and finally provide scientific and theoretical base for recoving rational structure oceanecological system and improving ecological condition of aquaculture pond. Resultsshowed:Seven associated heterotrophic bacteria, named Z-TG01, Z-TG02, Z-TG03,Z-TG04, Z-TG05, Z-TG06and Z-TG07, were isolated and purified from theco-cultural system using the method of plate coating, and Z-TG01, Z-TG02andZ-TG03were dominant associated heterotrophic bacteria, which the dominance indexbeing48.8%,14.0%and18.6%respectively. Using GN cards, three dominantassociated heterotrophic bacteria were identified Sphingomonas paucimobilis, Pantoea spp. and Myroides spp. respectively by the VITEK2Compact AutomaticBacteria Identification System (BioMérieux, France). Using Agrobacteriumtumefaciens KYC55, we found3dominant associated heterotrophic bacteria haveAHLs bioactivity, and increased gradually in the growth process of dominantassociated heterotrophic bacteria by measuring the activities of β-D-galactosidase.The supernatant of C. vulgaris culture in the exponential phases inhibited AHLsbioactivity of Z-TG01and Z-TG03significantly (p<0.01or p<0.05), and thesupernatant of C. vulgaris culture in the exponential phases inhibited AHLsbioactivity of Z-TG02significantly (p<0.01or p<0.05). C. vulgaris, which we used inpresent study, might secret analogs or quorum-quenching enzymes to inhibit AHLsbioactivity to interfere the release of toxins mediated by quorum sensing, and makeitself growthed in a suitable environment.The mutual action rule of C. vulgaris and dominant associated heterotrophicbacteria and the change rule of AHLs bioactivity of coculture system were studied inpresent study. The results showed:①Bacteria Z-TG01within a certain cell densityand C. vulgaris could promote mutual growth in co-culture system, and C. vulgaris ofthe exponential phases could obviously inhibited AHLs bioactivity of Z-TG01;②Bacteria Z-TG02of different cell density could inhibited the growth of C. vulgaris invarying degrees, and the AHLs bioactivity were not obviously inhibited, and the lowdensity of C. vulgaris or the instability of AHLs in the co-culture system of C.vulgaris and bacteria Z-TG02;③Bacteria Z-TG03within a certain cell density and C.vulgaris could promote mutual growth in co-culture system, and C. vulgaris of theexponential phases could obviously inhibited AHLs bioactivity of Z-TG03.Effects of AHLs (N-hexanoyl-DL-homoserine lactone, C6-HSL) on growth andantioxidant defense system of Chlorella vulgaris were studied in present study. Theresults showed that that low concentrations of C6-HSL (50,100and200nM) couldincrease the relative growth rate of C. vulgaris obviously at8h (p<0.05), higherconcentrations of C6-HSL (≥400nM) could decrease the relative growth rate of C.vulgaris obviously (p<0.05), and exhibited high concentration-dependent inhibition effects.50nM C6-HSL activated defense response of C. vulgaris in a short time (4h),increased activities of SOD, POD, CAT and GPX of C. vulgaris obviously (p<0.05),and decreased MDA content significantly (p<0.05). Compared to low concentrations,high concentration of C6-HSL (400nM) increased MDA content of C. vulgarissignificantly (p<0.05). The antioxidative enzymes of C. vulgaris increased to a peakthen decreased, and the MDA content of C. vulgaris increased significantly0to24hexposure to400nM C6-HSL (p<0.05).The mechanisms of C. vulgaris responding to AHLs were studied by iTRAQ(isobaric tags for relative and absolute quantification) at the protein expression level.The results showed that281proteins were identified,10proteins were differentiallyexpressed in C. vulgaris treated by400nmol/L C6-HSL,5were induced and5wererepressed. The exprsssion of photosystem II reaction center protein D1and heat shockprotein90was repressed, indicating the algal photosystem and other physiologicalfunctions had damaged in varying degrees. Meanwhile, the exprsssion of enolase,sedoheptulose-bisphosphatase, ATP synthase beta subunit and vacuolar ATP synthase,subunit A were significantly induced, indicating enhance anaerobic respiration andefficiency of ATP synthesis of the algal cell to provide energy for respairing thedamaged photosystem, the transmembrane transport of toxin, and normal growth.Moreover, the exprsssion of sulfolipid biosynthesis protein was significantly induced,indicating the algal cells were actively respairing the damaged cytomembrane.更多还原