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研究生：	陳羿甯 Chen, Yi-Ning
論文名稱：	白蝦Dscam 表現量、異構型歧異度與病原體耐受度之關聯性 Relationships among Dscam expression and diversity and pathogen tolerance in white shrimp
指導教授：	王涵青 Wang, Han-Ching
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 生物科技研究所 Institute of Biotechnology
論文出版年：	2014
畢業學年度：	102
語文別：	英文
論文頁數：	67
中文關鍵詞：	唐氏綜合症細胞黏附分子、特異性識別、白點症病毒
外文關鍵詞：	white shrimp, Dscam, Dscam isoform, WSSV
相關次數：	點閱：148 下載：5
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節肢動物Dscam屬於免疫球蛋白大家族中的一份子,參與免疫功能以及神經發育。典型的節肢動物Dscam具有10個免疫球蛋白結構域、6個第三型纖維黏接蛋白結構域、一個穿膜區以及胞質尾。然而在甲殼類動物中，發現除了典型Dscam外，也有不具胞質尾的Dscam存在。在結構上，Dscam於其胞外區域具有三個高度變異區，分別位在Ig2、Ig3以及Ig7，這些變異區的產生主要來自替代性RNA剪接機制，而因此產生多種Dscam異構型，並使得Dscam能夠辨認不同的病原體。因此本研究主要探討白蝦Dscam的表現量、異構型歧異度與對病原體耐受度的關聯性。本研究針對白蝦個體進行病毒感染前後Dscam表現量及異構型歧異度之分析，並從中了解其對蝦體病原體耐受度之重要性。因此首先針對16隻蝦體進行病毒感染前後Dscam的表現量以及感染後病毒帶原的偵測，並選定11隻蝦體進行Dscam異構型歧異度的分析，由此發現了病毒感染後Dscam異構型歧異度有降低的現象，並發現異構型歧異度的降低有助於增加蝦體對於病毒感染後的耐受性。藉由感染前後異構型種類的變化，我們篩選出一些可能對WSSV有結合專一性的Dscam異構型。

Arthropod Dscam belongs to the Ig superfamily. The protein plays a variety of roles in the immune system and also participates in neural guidance. The typical structure of Dscam consists of 10 Ig domains, six fibronectin type III (FN) repeats, a transmembrane (TM) domain, and a cytoplasmic tail. A unique tail-less Dscam (lacking either a TM domain or a cytoplasmic tail) has also been identified in crustaceans. Hypervariability in the Dscam extracellular region (due to alternative splicing in the Ig2, Ig3, and Ig7 regions) appears to enable the recognition of numerous pathogens. The objective of this study was to identify relationships among Dscam expression, the resulting diversity of Dscam isoforms, and their ability to fight pathogens. Individual shrimp were tagged and divided into two groups: those injected with PBS (control) and those injected with white spot syndrome virus (WSSV). Hemocyte samples were collected pre-infection and at 48 hpi. Real-time PCR was used to detect the expression of Dscam and WSSV VP28, whereupon Dscam Ig2 and Ig3 regions were amplified. Based on the Dscam response in individual shrimp (sampled before and after viral infection), we concluded that a decrease in Dscam variability may render shrimp less susceptible to infection with WSSV. In addition, we identified specific Dscam isoforms seem to show an ability against WSSV.

Table of contents
中文摘要(Chinese abstract)............................. I
Abstract............................................. II
Acknowledgements..................................... III
Table of Contents ................................... IV
Table Index ......................................... VII
Figure Index ........................................ VIII
Abbreviation ........................................ IX
1. Introduction ..................................... 1
1-1 The immune systems of invertebrates ............. 1
1-2 The structure of arthropod Dscam................. 2
1-3 The functions of arthropod Dscam ................ 4
1-4 The aims of this study .......................... 5
2. Materials and Methods ............................ 7
2-1 Experimental shrimp ............................. 7
2-2 Total RNA extraction ............................ 7
2-3 Reverse transcription and cDNA synthesis ........ 8
2-4 Quantitative real-time PCR ...................... 8
2-5 Polymerase chain reaction (PCR).................. 9
2-6 Purification of PCR products .................... 10
2-7 Cloning and blue and white screening ............ 10
2-8 Analysis of variability in Dscam variable Ig2 and Ig3 ..................................................... 12
2-9 Statistical analysis ............................ 12
2-10 Construction of recombinant elite Dscams ....... 13
2-11 Expression of recombinant elite Dscams ......... 13
2-12 SDS poly-acrylamide-gel-electrophoresis and western blot analysis ....................................... 15
3. Results .......................................... 17
3-1 Expression of Dscam increased 48 hours post WSSV infection ........................................... 17
3-2 Shrimp Dscam may help defend against WSSV in a quantity-independent manner .................................. 17
3-3 High Dscam expression in shrimp with persistent WSSV infection may render them increasingly susceptible to WSSV infection under immune stimulation .................. 18
3-4 Decrease in Dscam diversity as a response to WSSV infection............................................ 19
3-5 Shrimp with relatively high diversity in WSSV-induced Dscam isoforms appear more resistant to WSSV ........ 20
3-6 WSSV-induced formation of elite Dscam isoforms in WSSV-infected shrimp at 0 hpi and 48 hpi ................. 21
3-7 Shrimp with low WSSV scores had higher proportions of WSSV-induced elite Dscam isoforms ................... 22
3-8 The production and expression of 10 elite Dscams and 2 control Dscams ...................................... 22
4. Discussion ....................................... 24
5. References ....................................... 30

Table Index
Table 1. Primers used in this study ................. 36
Table 2. Dscam diversity of each shrimp ............. 37
Table 3. Dscam isoforms with corresponded plasmid ... 40
Table 4. Summary of elite Dscams .................... 50
Table 5. List of constructed elite Dscams with corresponded Dscam isoforms ...................................... 51

Figure Index
Figure 1. Expression of Dscam in hemocytes from individual shrimp before (0 hpi) and after (48 hpi) PBS or WSSV injection ........................................... 52
Figure 2. Relationships between the expression of Dscam and VP28 in shrimp injected with PBS and relationships between the expression of Dscam, ProPO, and VP28 in shrimp injected with WSSV ........................................... 54
Figure 3. Complexity of Dscam Ig2 and Ig3 regions in shrimp injected with PBS (control) or WSSV ................. 57
Figure 4. Relationship between combinations of Dscam isoforms and WSSV scores (with severity determined according to VP28 expression) ................................. 59
Figure 5. Distribution of super and WSSV-induced elite Dscam Ig2 and Ig3 in shrimp with low, medium, and high levels of VP28 ................................................ 61
Figure 6. Alignments of multiple amino acid sequences associated with WSSV-induced elite Dscam Ig2 and Ig3 ..................................................... 63
Figure 7. Occurrence of Dscam Ig2 and Ig3 before and after injection with PBS or WSSV .......................... 64
Figure 8. Expression of 10 recombinant elite Dscams and 2 non-elite Dscams .................................... 66

                                    

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