利用遺傳工程與基因轉殖的技術以促進石斑魚魚苗的快速成長

Abstract

石斑魚是世界性的養殖魚種，特別是在亞洲地區漁撈量約佔全世界86％，在世界糧農組織統計（FAO statistics, 2007），至目前為止，全世界的石斑魚生產量約二十三萬二千頓，但仍不敷整體市場需求。因此石斑魚的水產養殖也受到重視，而這個產業重要的關鍵又在於魚苗的量產技術。因此，在本研究欲利用基因轉殖與遺傳工程的方式，來增加石斑魚魚苗的生長速度及存活率。本論文共分為兩章節，首先第一章是建立基因轉殖豐年蝦做為生物反應器來生產外源性蛋白。也就是將帶有由human cytomegalovirus及medaka β-actin的 promoter驅動黃鰭鯛（yellowfin porgy; Acanthopagrus latus）生長激素（ypGH） cDNA之質體及驅動綠色螢光（GFP）報導基因之質體共同轉殖至野生種豐年蝦（Artemia sinica）中，藉由報導基因的表現，在螢光顯微鏡下GFP，由具有綠色螢光的豐年蝦中篩選得到含有ypGH基因的豐年蝦(ypGH-Artemia)，經繼代遺傳以雜交方式篩選出穩定的轉殖豐年蝦品系A3及A8。經由酵素連結免疫吸附法（enzyme-linked immunosorbent assay）檢測出每50隻同型合子A3 及A8之ypGH-Aremia分別含有0.089 and 0.032 μg的ypGH。接著將A3之ypGH-Aremia餵食斑馬魚（Danio rerio）魚苗，證明可以促進斑馬魚魚苗體長比對照組高出16-20％。第二章是利用餵食ypGH-Aremia與浸泡rGH的方式促進石斑魚魚苗生長。我們將ypGH-Aremia投餵石斑魚（Epinephelus coioides）魚苗。歷經25天後結果顯示餵食ypGH-Aremia的實驗組可促進石斑魚魚苗之生長，例如增重率比對照組高出640％，體重高出約62％，體長高出19％。另一方面，我們利用從大腸桿菌大量生產經過純化後的重組ypGH（rGH）以浸泡石斑魚（E. malabaricus）魚苗，實驗進行17天後，結果顯示以濃度500 μg/liter浸泡30分鐘可讓增重率比對照組高出170％，體重高出約69％，效果最好。總結，本實驗證實了餵食具有ypGH的基因轉殖豐年蝦及浸泡E. coli表現系統生產具生物活性的rGH，都具有促進石斑魚苗生長的效果。這結果也將提供了利用分子生物技術的方法以增進石斑魚魚苗產量的可行性。

Grouper is a world-wide aquaculture fish. Particularly around 86％ of fishing catch of grouper is in Asia. According to FAO statistics, so far, the world's grouper catch is about 232,000 tons, but still fall short of the overall market demand. The industrial production of grouper is overwhelmingly interested in marine aquaculture. Yet, the technology of mass production of grouper fry is critical for grouper industry. The main goal of this study is to enhance the growth rate and survival rate of grouper fry using gene transferring and genetic engineering. In this dissertation, I described two my study issues in two chapters. Chapter one is “Development of transgenic zooplankton Artemia as a bioreactor to produce exogenous protein”. Two expression plasmids were co-electroporated on decapsulated cysts of wild type Artemia sinica: pCMV-Mβ-ypGH contained yellowfin porgy GH (ypGH) cDNA and pCMV-Mβ-GFP contained GFP reporter gene. I examined the GFP shown in the Artemia larvae by fluorescence microscopy and found two stable ypGH transgenic lines (ypGH-Artemia) named A3 and A8. I examined recombinant ypGH produced by ypGH-Artemia through enzyme-linked immunosorbent assay and found that the concentration of 0.089 and 0.032 μg ypGH per 50 homozygotic nauplii, respectively. The average body length gain rates of zebrafish (Danio rerio) larvae fed A3 strain of ypGH-Artemia were 16–20% greater than those of control group. Chapter two is “Growth enhancement of grouper fry by feeding transgenic Artemia containing growth hormone cDNA and by immersion recombinant growth hormone”. I fed grouper (Epinephelus coioides) larvae with live ypGH-Artemia. At each feeding, grouper larvae were given three to four ypGH-Artemia four times a day for 25 days. Results showed that feeding grouper with ypGH-Artemia enhanced the growth of grouper larvae most prominently (P<0.01). Compared with the control group, the weight gain rate and average body weight of GH-Artemia-consumed experimental larvae was 640% and 62% higher than those of Artemia-consumed control larvae, respectively. The average body length of experimental larvae was 19% higher than that of control larvae. Furthermore, recombinant ypGH (rGH) produced by Escherichia coli expression system was purified. Biological activity of rGH was assayed by immersing grouper (E. malabaricus) fries in seawater containing rGH. After 17 days of experiment, results showed prominent growth enhancement by immersing grouper fry with 500 μg rGH/liter for 30 min. The weight gain rate and average body weight of treated fry was 170% and 69% higher than those of the control fry, respectively, suggesting the rGH is functional for growth of grouper fry. The line of evidences, I concluded that either feeding ypGH-Artemia or immerse rGH can greatly enhance the growth of grouper larvae and fry, which might be highly potentially useful in aquaculture of grouper.