本研究探討白蝦 Litopenaeus vannamei九種免疫相關基因的組織表現以及受口服葡聚多醣的調控。這九種基因為葡聚多醣結合蛋白-高密度脂蛋白((β-glucan binding protein-high density lipoprotein, BGBP-HDL)、脂多醣/葡聚多醣結合蛋白(lipopolysaccharide/β-glucan binding protein, LGBP)、血藍素 (hemocyanin)、原酚氧化酶(prophenoloxidase, proPO)、轉醯胺酶(transglutaminase, TGase)、對蝦素(penaeidin-3, PEN-3)、crustin, 細胞質錳型超氧歧化酶(cytosolic manganese superoxide dismutase, cMnSOD)、和溶菌酶(lysozyme)。研究包括(1) 白蝦proPO 及LGBP的cDNA選殖及表現特性分析；(2)九種基因在大蝦時期的組織表現；(3) 九種基因在蝦苗時期的表現和 (4) 口服葡聚多醣對九種基因表現的調控。
cDNA序列選殖及表現特性分析研究結果顯示proPO基因全長2061 bp，轉譯686個胺基酸。其蛋白質序列與草蝦和熊蝦有85%的高相似性，與其他甲殼類的相同度在58%到77%之間。北方墨點分析結果顯示proPO主要在血細胞內表現，藉由反轉錄聚合酶連鎖反應(RT-PCR)可在多處組織偵測到proPO的表現。原位雜交結果顯示proPO主要表現在浸潤於各器官組織間的血球。LGBP基因全長為1104 bp，轉譯367個胺基酸，包括17個訊息胜肽。其蛋白質序列與藍蝦相似性最高(97%)，與草蝦和大明蝦的相似性也在90%以上，與其他甲殼類相同度在63%到68%之間。北方墨點分析結果顯示LGBP主要在肝胰臟表現，原位雜交結果顯示表現LGBP的主要是肝胰臟F細胞。
以proPO和LGBP全段cDNA及其他七種免疫相關基因部分片段，用RT-PCR、即時定量PCR(qPCR)和原位雜交分析免疫基因在白蝦的組織表現情形，結果顯示BGBP-HDL、LGBP和血藍素主要表現於肝胰臟，其表現量為β-actin的10 – 30%。proPO、TGase、crustins、PEN-3和溶菌酶的最高表現量均在血球，而在肝胰臟最低。他們在血球內的表現程度約為β-actin的3倍(PEN-3)到10-2倍(proPO)之間。cMnSOD最高表現量則出現在血液相關的組織。原位雜交結果顯示表現BGBP-HDL、LGBP和血藍素主要的細胞型態是肝胰臟F細胞，而proPO、TGase、crustins、PEN-3和溶菌酶則表現在浸潤於組織的血球細胞。肝胰臟F細胞和血球細胞都會表現cMnSOD。
以qPCR和原位雜交分析胚胎到後期幼生(postlarvae, PL)各階段白蝦九種基因的表現情形，結果顯示除了proPO和血藍素沒有在胚胎及蝦苗前期階段表現之外，其他基因表現在所有檢測的時期均可利用qPCR偵測到。除了BGBP-HDL和cMnSOD在胚胎及蝦苗前期表現情形明顯較眼幼蟲期、糠蝦期和PL初期高之外，其他基因表現量均以胚胎及蝦苗前期最低。九種基因表現量最高均出現在PL20。使用原位雜交技術，BGBP-HDL、LGBP 和SOD的mRNA出現在早期蝦苗階段的管狀構造，推測這些構造可能為肝胰臟和消化道的前身，而crustin和TGase的表現則可在糠蝦期階段被偵測出。所有的基因表現在PL階段均可觀察到，BGBP-HDL、LGBP、SOD和血藍素主要表現在肝胰臟而BGBP-HDL和SOD的表現還可在神經節發現。
利用qPCR分析白蝦大蝦及PL階段口服葡聚多醣對這九種基因表現的調控，發現每天餵食含葡聚多醣的飼料七天，葡聚多醣的劑量2或10 g kg-1並沒有顯著影響各基因的表現。BGBP-HDL和LGBP的基因表現在口服葡聚多醣後有上升趨勢，到第七天達到顯著差異。溶菌酶和cMnSOD的表現則分別在6 h和12 h顯著增加，並到24 h和72 h時減少且恢復到原本的表現量。PEN-3的表現則在6 h顯著下降而24 h時恢復。Crustin的表現與PEN-3有類似的趨勢但是沒有顯著差異。血藍素、proPO和TGase的表現則不受口服葡聚多醣的調控。後期幼生白蝦BGBP-HDL的基因表現在口服葡聚多醣後6 h即有顯著上升，而TGase的表現則在12 h顯著增加並到第七天回復。另一方面，LGBP、血藍素、溶菌酶和crustin的表現先呈顯著下降，而血藍素於12 h即恢復，溶菌酶24 h恢復，而LGBP和crustin則持續到7d還未恢復。
整體而言， 白蝦的免疫相關基因在平時即有固定表現，但各基因表現程度各有不同，主要的表現位置為肝胰臟或血球。口服會影響基因的轉錄，而顯示葡聚多醣處理後所引起的抗病能力增強是由許多不同的機制共同造成的現象，其中包括部分免疫基因mRNA表現的提昇。免疫基因發生學的研究顯示，多數免疫基因在蝦苗發育早期即有表現，意涵利用免疫刺激物處理蝦苗期之白蝦應是可行的，而這些免疫基因在發生學上的表現同時也成為評估蝦苗時期免疫能力的有效工具。

The present study investigated the expression profiles of nine genes involved in immune defense of the Pacific white shrimp Litopenaeus vannamei and their responses to oral administration of beta-1,3-glucan. The nine immune related genes were beta-glucan binding protein-high density lipoprotein (BGBP-HDL), lipopolysaccharide/beta-glucan binding protein (LGBP), hemocyanin, prophenoloxidase (proPO), transglutaminase (TGase), penaeidin-3 (PEN-3), crustin, cytosolic manganese superoxide dismutase (cMnSOD), and lysozyme. A series of experiments were carried out in the study including: (1) cDNA cloning and characterization of proPO and LGBP; (2) tissue mRNA expressions of the nine genes in adult shrimp; (3) expression and localization of the nine genes during larval and postlarval ontogenic development; (4) the effects of dietary beta-1,3-glucan on the expression of the nine genes.

The cDNA cloning study showed that the proPO cDNA contains an open reading frame of 2061 bp and encodes a 686 amino-acid peptide. The protein sequence of the proPO has a similarity of 85% with those of Penaeus monodon and P. semisulcatus and has an identity of between 58 and 77% with other crustaceans. Northern blot analysis revealed that proPO was constitutively expressed mainly in hemocytes. Its transcripts were observed in hemocytes and many other tissues when detected with RT-PCR. The results of in situ hybridizations showed that the hemocytes that infiltrated in tissues were responsible for the positive signals. The LGBP cDNA contains an open reading frame of 1104 bp and encodes a 367 amino-acid protein with a 17 a. a. signal peptide. The protein sequence of the LGBP has a similarity of 97% with LGBP of L. stylirostris, >90% identity with BGBP of P. monodon and LGBP of Fenneropenaeus chinensis and has an identity of between 63 and 86% with other crustaceans. Northern blot analysis revealed that LGBP was constitutively expressed mainly in hepatopancreas. The results of in situ hybridizations showed that the hepatopancreatic F cells might be the major cell type for LGBP production.

Using the complete cDNAs of proPO and LGBP and partial fragments of the other seven genes, their tissue expressions were analyzed by conventional RT-PCR, quantitative real time PCR (qPCR) and in situ hybridization. The results demonstrated that BGBP-HDL, LGBP and hemocyanin were mainly expressed in the hepatopancreas and their expressions levels were about 10 to 30% those of

TABLE OF CONTENTS

CHAPTER 1 GENERAL INTRODUCTION…. 1
CHAPTER 2 GENERAL MATERIALS AND METHODS…31
CHAPTER 3 GENE CLONING AND CHARACTERISATION OF PROPHENOLOXIDASE AND LIPOPOLYSACCHARIDE/

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