澳洲球形海綿(Cinachyrella australiensis (Demospongiae: Spirophorida))廣泛分布於西太平洋沿岸、印度洋、澳洲沿岸。在台灣恆春萬里桐潮間帶潮池中可發現其蹤跡。在先前研究發現此海綿含有疑似硫氧化共生菌存在。澳洲球形海綿可以出芽方式行無性生殖，芽體如何獲得母體之共生菌則尚待釐清。本研究從成體海綿取芽體萃取其DNA，以細菌共通16S rDNA基因引子對，以PCR方式增幅並純化其DNA片段後再經基因選殖並隨機挑選20個菌落針對16S rDNA進行定序。同時，成體海綿也以同樣方式處理。結果顯示，成體海綿中最優勢共生菌之出現頻率為65%，而此優勢共生菌在芽體出現之頻率為15%。此共生菌含有RubisCO基因與深海管蟲硫氧化關係密切是一自營性細菌共生菌。利用螢光原位雜合法(FISH)觀察共生菌存在海綿成體的皮質層細胞、中質層的原始生殖細胞及芽體的細胞中之分布。在芽體形成過程是由成體海綿表面隆起發育而成，在此交界區域(含芽體部分)之細胞以螢光原位雜合法證實富含優勢共生菌，顯示成體海綿是透過垂直傳遞的方式將共生菌傳遞與其芽體，除了最優勢的共生菌外，海綿細胞內其他共生菌其中一部分亦被證實是自營性細菌。

The sponge Cinachyrella australiensis (Demospongiae: Spirophorida) is widely distributed in Indian ocean, West Pacific ocean, and Australian waters. It also can be found in the intertidal pools of Wun-Li-Ton in southern Taiwan. The sponge can propagate asexually by budding. According to the previous studies, this sponge was suspected to be symbiotic with sulfur-oxidizing chemoautotrophic bacteria. How the generation do obtain this symbiont is still unknow. In this study, PCR was used to amplify the DNA extracted from buds and sponges to obtained the 16S rDNAs. A total of 20 clones from each bud and mature sponge samples were randomly selected and sequenced. The results indicated that the major symbiotic bacteria constitute 65％ of the clones derived form the mature sponge and 15％ from the buds. The dominant symbionts contain RubisCO gene and are related to the sulfur-oxidizing chemoautotrophic bacteria, associated with the tube worms of the deep sea hydrothermal vents. The location of the sulfur-oxidizing chemoautotrophic bacteria was observed by fluorescence in situ hybridization (FISH). It was found that the sulfur-oxidizing chemoautotrophic bacteria were intracellular symbiosis within the cells of cortex, archaeocytes of mesoglial, and bud. Similar results were also observed at the junction of a developing bud and mature sponge. Apparently, the symbionts are transmitted from sponge to bud vertically. Furthermore, in this study, we also found several other intracelluar symbionts besides the major symbiotic bacterium,some of them are autotrophic in nature.

一、前言..................................................4
二、材料與方法............................................8
三、結果.................................................19
四、討論.................................................32
五、參考文獻.............................................40

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