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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48623

Title: 軟絲與細菌的共生關係:卵巢發育和副纏卵腺中共生菌的族群結構變化之探討
The symbiotic relationship between bacteria and oval squid, Sepioteuthis lessoniana: The research of the ovarian development and the bacterial community variation in the accessory nidamental glands
Authors: Chen, Chih
陳稚
Contributors: NTOU:Department of Aquaculture
國立臺灣海洋大學:水產養殖學系
Keywords: 大分子脂質運輸蛋白;轉錄體;總體基因體學
Large lipid transfer protein;Transcriptome;Amplicon analysis
Date: 2017
Issue Date: 2018-08-22T06:06:47Z
Abstract: 萊氏擬烏賊(Sepioteuthis lessoniana,俗稱軟絲)的副纏卵腺(accessory nidamental gland, ANG)是卵巢的附屬生殖腺體,副纏卵腺內共生菌的生長與卵巢發育相關,成熟時因其內共生菌的色素沉澱而變為紅色或黃色。然而副纏卵腺的發育在組織學上沒有顯著的特異性因此本研究擬以卵巢發育為輔,瞭解宿主(副纏卵腺)發育與寄主(共生菌)組成結構的關係,並探討宿主對共生菌的可能篩選或調控方式。本研究分成兩部分,第一部分是探討卵巢發育及相關基因的功能分析,第二部分則是探討副纏卵腺發育期間的菌群結構變化及差異表現基因。於第一部分首先以組織學建立卵巢發育階段,接著從卵巢的轉錄體(Transcriptome)中搜尋可能可作為卵巢發育標記或調控卵巢發育的基因,包括濾泡細胞增生、性類固醇合成酶、性類固醇受器和卵黃堆積等相關基因,並透過qPCR分析這些基因的表現模式,並嘗試建立軟絲的組織培養技術。與第二部分也首先以組織學建立副纏卵腺發育階段,再利用總體基因體(Amplicon analysis)分析副纏卵腺發育階段的菌群變化,接著利用不同發育階段的轉錄體搜尋在副纏卵腺中具有差異表現的基因。由組織學結果顯示,當卵巢中有濾泡細胞增生現象時,副纏卵腺中開始有菌的出現,當卵巢中有卵黃堆積期卵時,副纏卵腺中開始有共生菌產生色素沉澱的現象。由基因表現分析結果顯示軟絲的第二型骨塑型蛋白及其第1B型受器(bone morphogenetic protein type II, bmp2和bone morphogenetic protein receptortype-1B, bmpr1b)與濾泡細胞增生高度相關,卵黃素(vitellogenin 1, vtg1和vitellogenin 2, vtg2)則與卵黃堆積高度相關,而軟絲的載脂蛋白(apolipophorinlong isoform, apoLp-long)在成熟濾泡期會高量表現在濾泡細胞上,因此推測這兩個基因與最終成熟相關之外,可能為卵巢用來調控副纏卵腺發育(如促使共生菌的色素沉澱)的機制。總體基因體分析結果顯示,隨著發育成熟,副纏卵腺中的Alphaproteobacteria比例有變多的趨勢,但在104~105年期間和105~106年期間所被篩選的菌種卻不相同,分別為Alphaproteobacteriaunclassified及Rhodobacteria,因此推測副纏卵腺並非對特定菌種具有專一性,而對細菌的某一功能具有需求。副纏卵腺的轉錄體中具有差異表現的基因多與代謝、運輸相關,但沒有與抗菌肽相關的基因,因此推估副纏卵腺可能是利用營養或離子的微環境限制來抑制細菌的生長。綜合上述,本研究結果指出ANG發育和卵巢發育高度相關,且共生菌的族群會被宿主所篩選,而為了瞭解宿主與共生菌之間溝通機制,我們需進一步去探討共生菌的生物特徵。
Accessory nidamental glands (ANG) is the accessory reproductive gland of female in Sepioteuthis lessoniana (oval squid). Symbiotic bacteria growth is associated with ovarian development and ANG contain colorful pigments in mature female. The objective of this study was to investigate that the communication between the host (ANG) and symbiotic bacteria during ovary growth. However it is difficult to identify ANG development by histology. To solve this problem, the ovary developmental stage was used as an assistance. To find the marker genes for ovarian development stages, ovarian transcriptomic data was used to select candidate genes. Real time PCR (qPCR) was used to confirm the gene expression profiles in different ovarian stages. To understand the bacterial selection in the ANG (host), amplicon analysis was used to classified the variety of bacterial community structure during ANG development. Furthermore, transcriptomic analysis was used to understand the molecular mechanism of ANG-bacteria communication by ANG. According to the histological data, the bacteria clonies were observed in the ANG while the follicle cells penetrated into the oocytes. The colonies of symbionts were observed the yellow and red pigments in the ANGs while oocytes entered vitellogenic stage. Based on the gene expression profiles (qPCR analysis), several genes was selected as the marker for different ovary stages. These genes include bmp2 (bone morphogenetic protein type II) and bmpr1b (bone morphogenetic protein receptor type-1B) as the folliculogenetic marker), vtg1 and vtg2 (vitellogenin type 1 and type 2) as the vitellogenetic marker, apoLp-long and apoLp-short (apolipophorin long isoform and short isoform) as the mature follicle (before ovulation) marker. According to amplicon analysis, the bacterial population are strongly selected by ANG. However, the dominant bacteria showed defference in the different collection periods (year 104~105: Alphaproteobacteria unclassified, year 105~106: Rhodobacteria). Taking together, our datas demonstrates that ANG developmental stage are highly associated to ovarian growth. The bacteria population are selected by host. To clarify the mechanism of host-bacteria communication, we need to identify the characteristics of target bacteria in next step.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010433027.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48623
Appears in Collections:[水產養殖學系] 博碩士論文

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