臺灣博碩士論文加值系統

摘要

原酚氧化酵素(prophenoloxidase, proPO)及超氧歧化酵素(superoxide dismutase, SOD)為甲殼類重要的免疫相關基因，本篇研究從澳洲螯蝦 (Cherax quadricarinatus)血球中選殖出上述基因，並人為感染白點症病毒 (white spot syndrome virus, WSSV)或親水性產氣單胞菌 (Aeromonas hydrophila)，分析澳洲螯蝦之存活率、總血球數、基因表現及酵素活性。應用半定量RT-PCR (semiquantitative RT-PCR)及RACE (rapid amplification of cDNA ends)兩種技術，從血球細胞cDNA pool中選殖出proPO及cMnSOD (cytosolic manganese superoxide dismutase)基因之cDNA序列全長。proPO的cDNA序列全長為2,930 bp，最長的開讀框 (open reading frame)轉譯出的氨基酸序列全長為665 aa，預測的分子量為75.8 kDa，等電點為6.08。cMnSOD的cDNA序列全長為1,096 bp，最長的開讀框轉譯出的氨基酸序列全長為287 aa，預測的分子量為31.3 kDa，等電點為5.82。proPO和cMnSOD與已知序列的親緣分析，澳洲螯蝦的proPO和通訊螯蝦 (Pacifastacus leniusculus)及美國螯蝦 (Procambarus clarkia)最為接近，澳洲螯蝦的cMnSOD則和美國螯蝦 (Procambarus clarkia)最為接近。proPO及cMnSOD基因的組織表現分布，在血球、肝胰腺、心臟、鰓、生殖腺、消化道和肌肉中皆有存在。其中proPO在血球、心臟及鰓的表現較為明顯，cMnSOD於血球、心臟、肝胰腺及鰓的表現較為明顯。人為感染之存活率結果顯示，感染白點症病毒會造成100%的死亡，親水性產氣單胞菌則不會造成螯蝦死亡，並在6小時內清除血淋巴的細菌。總血球數分析顯示，相較於注射PBS，感染白點症病毒的澳洲螯蝦總血球數在第48、72小時明顯的下降，感染親水性產氣單胞菌的澳洲螯蝦總血球數僅在第24小時下降，於48及72小時便已恢復。人為感染白點症病毒，澳洲螯蝦proPO基因相對表現量除了第24小時有顯著上升，在第6、12、48、72小時呈現顯著下降，人為感染親水性產氣單胞菌，澳洲螯蝦proPO基因相對表現量在第12、24小時呈現出顯著上升。而單位血球中所含有的PO活性，相較於PBS組，皆在第24、48、72小時顯著上升 (不論感染何種病原體)。人為感染白點症病毒，澳洲螯蝦cMnSOD基因相對表現量除了第24小時顯著上升，在第6、12、48、72小時呈現顯著下降，人為感染親水性產氣單胞菌，澳洲螯蝦cMnSOD基因相對表現量在第12、24小時呈現顯著上升。而單位血球中所含有的SOD活性，皆在第48、72小時顯著上升 (不論感染何種病原體)。

Abstract
Prophenoloxidase (proPO) and cytosolic manganese superoxide dismutase (cMnSOD) play crucial roles in crustacean innate immunity. In the present study, both of above genes were cloned from hemocytes of the red claw crayfish Cherax quadricarinatus using reverse transcription- polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) method, and then total hemocyte counts (THCs), gene expression and enzyme activities were characterized after injection of white spot syndrome virus (WSSV) or Aeromonas hydrophila. The full length cDNA of proPO is 2930 bp and encodes 665 amino acids, wherease the cMnSOD cDNA is 1096 bp in length and encodes 287 amino acids. The calculated molecular mass of translated protein of proPO and cMnSOD is 75.8 kDa and 31.3 kDa with an estimated pI of 6.08 and 5.82, respectively. Phylogenetic analysis of amino acid sequences showed that C. quadricarinatus proPO and cMnSOD are more closely related to the proPO and cMnSOD of other crayfish than to that of penaeids, crabs, lobsters, or freshwater prawns. Tissue distributation analysis revealed that the proPO is primarily expressed in hemocytes, gill and heart, though the higher expression levels of cMnSOD showed in hemocytes, gill and hepatopancrease. The crayfish artificially infected with WSSV all died within 4 days generally. However, there was no mortality in crayfish when infected with A. hydrophila. THCs significantly decreased in crayfish at 48 h and 72 h after the infection of WSSV compared to the PBS group. In contrast, THCs of crayfish after A. hydrophila challenge were recovered at 48 h and 72 h from the significantly lower level at 24 h. The expression level of proPO and cMnSOD transcripts in hemocytes varied greatly after the challenge of WSSV or A. hydrophila. After WSSV challenge, the expression of both genes were significantly up-regulated at 24 h, but down-regraulated at other time points including 6 h, 12 h, 48 h and 72 h. While after the infection of A. hydrophila, both genes transcripts only significantly increased at 12 h and 24 h compared with the PBS group. There were similiar responses for the enzyme activity toward WSSV and A. hydrophila infection. The PO activities per hemocyte significantly increased from 24 h to 72 h compared to the PBS group, and cMnSOD exhibited higher level later at 48 h and 72 h post virus and bacteria infection.

【目錄】
1.前言………………………………………………………………1
1.1甲殼類免疫……………………………………………1
1.2原酚氧化酵素 (prophenoloxidase, proPO)………………1
1.3胞內錳型超氧歧化酵素 (cytosolic manganese superoxide
dismutase, cMnSOD)…………………………………3
1.4白點症病毒 (white spot syndrome virus, WSSV)................4
1.5親水性產氣單胞菌(Aeromonas hydrophila).........................5
1.6澳洲螯蝦 (Cherax quadricarinatus).……..………………..6
1.7實驗目的...................................................……………….....6
2.材料與方法.......................................................................................8
2.1實驗動物…………………………………………………....8
2.2 RNA萃取與cDNA合成…………………………………..8
2.3基因選殖 (proPO, cMnSOD)……………………………...9
2.4序列分析與親緣分析……………………………………...9
2.5組織表現分析……………………………………………..10
2.6澳洲螯蝦人為感染白點症病毒…………………………..11
2.7澳洲螯蝦人為感染親水性產氣單胞菌…………………..11
2.8 WSSV感染存活率分析….……………………………….12

2.9細菌清除分析..……………………..…………………….12
2.10總血球數分析…………………………………………...13
2.11基因表現分析…………………………………………...13
2.12酚氧化酵素活性分析…………………………………...14
2.13超氧歧化酵素活性分析………………………………...15
2.14統計分析………………………………………………...17
3.結果……………………………………………………………....18
3.1原酚氧化酵素及胞內錳型超氧歧化酵素之基因選殖
與序列分析………….…………………………………...18
3.2原酚氧化酵素及胞內錳型超氧歧化酵素
之親緣分析.……………………………………………...19
3.3原酚氧化酵素及胞內錳型超氧歧化酵素在澳洲螯蝦
不同組織的基因表現情形……………….……………...20
3.4澳洲螯蝦人為感染白點症病毒後之存活率…………....20
3.5細菌清除分析……………………………………………21
3.6總血球數分析…………………………….……………...21
3.7原酚氧化酵素基因表現及酚氧化酵素活性分析..…….22

3.8胞內錳型超氧歧化酵素基因表現及超氧歧化酵素
活性分析…….....................................................................22
4討論.….…………………………………………………………..24
5. 圖&表….………………………………………………………..31
參考文獻…………………………………………………………...50

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