利用分子生物技術改良水產生物品系

Improve Aquatic Strains by Using Molecular Biotechnology

李思賢(Si-Shen Li)

指導教授 ： 蔡懷楨

國立臺灣大學/生命科學院/分子與細胞生物學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.10241

摘要

為了改良水產生物品系，本研究利用分子生物技術開發甲殼類與微細藻的基因轉殖系統，並希望藉此提升水產養殖的競爭力。淡水長臂大蝦(Macrobrachium rosenbergii)是台灣水產養殖的重要物種，也是水產無脊椎中重要的模式動物。本研究開發出精荚顯微注射法[spermatophore microinjection(SMI)]針對此一物種進行基因轉殖，並成功獲得基因轉殖品系。實驗證明將濃度範圍從28 到1,000 ng/ml的環狀質體(pGL)以SMI方式注射到精荚中並經體內授精與孵化後，轉殖組與控制組在授精率及孵化率上並無顯著差異。再以PCR方式檢測授精二十一天的蝦苗的體染色體，發現在實驗組中有一分子量與正控制組相同(680 bp)且只會在轉殖組產生的DNA序列被放大出來的DNA片段，南方點墨法的實驗結果也證實此段DNA片段就是所轉殖的外源性基因。PCR與南方點墨法的實驗結果顯示，以濃度為750ng/ml的DNA溶液進行SMI時可以達到 70% (16/23)的轉殖率，進一步的實驗結果也顯示以SMI進行基因轉殖其轉殖率與所用的DNA濃度呈正相關。上述實驗結果清楚的顯示出SMI是目前淡水長臂大蝦進行大量基因轉殖時最簡單且高效率的基因改造技術。在另一方面，微細藻中的擬球藻(Nannochloropsis oculata)是目前常用於海水水產養殖的重要物種。我利用基因轉殖技術創造出了能生產牛抗菌蛋白[bovine lactoferricin (LFB)]的基因轉殖擬球藻。我將已針對微細藻最佳化的LFB cDNA與紅螢光報導基因(DsRed)融合後，與來自萊因綠藻(Chlamydomonas reinhardtii)的熱誘導啟動子組合(heat shock protein 70A promoter + ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit 2’ promoter)構築成微細藻的抗菌蛋白表現殖體，並利用電穿孔法將其送入擬球藻。在491株藻株中我篩選到兩株能穩定保有外源性基因達22個月以上的轉殖株，且在熱誘導處理後的轉殖株能在RT-PCR的實驗中證實LFB-DsRed gene能在轉殖藻株中進行轉錄。蛋白質的分析結果也顯示轉殖藻株能產生可被anti-DsRed 單株抗體所辨識的LFB-DsRed蛋白質。這些經過熱誘導的轉殖藻株以口服注射(1×108 cells/ per fish)的方式投餵到日本稻田魚(Oryzius laptipes)後，在經過六小時的消化後以腸炎弧菌(Vibrio parahaemolyticu)進行感染實驗，其二十四小時存活率比口服注射同劑量野生擬球藻的控制組高[91.6% (n=12) versus 0% (n=6)]。此一結果顯示投餵含有LFB的基因轉殖擬球藻，能夠保護日本稻田魚不因腸炎弧菌的感染而死亡。

關鍵字

水產生物 ； 分子生物技術 ； 基因轉殖

並列摘要

In this study, I developed the transgenic systems on shellfish and microalga in order to improve the quality of aquaculture organisms. A spermatophore microinjection (SMI) technique was developed allowing exogenous DNA fragments to be transferred easily into Macrobrachium rosenbergii, an important aquacultural shellfish and aquatic invertebrate model. From 28 to 1,000 ng of the circular plasmid pGL, in a total volume of 1 ml, were directly microinjected into spermatophores. Fertilization and hatching of prawns created with SMI were completed in vivo. Fertilization and hatching rates in the SMI treatments did not differ from those of the untreated control group. The genomes of free swimming, SMI-created larvae (21 days after fertilization) were analyzed using PCR and Southern blot analyses. A product with a molecular mass of 680 bp was amplified. It corresponded to amplifications of pGL, and Southern blot analysis revealed that the amplified band was positive. The gene transfer rate was primarily dependent on the concentration of DNA during SMI. The higher the concentration of pGL, the higher the rate of gene transfer. PCR and Southern blot analyses detected the existence of foreign DNA in 16 of 23 samples (70%) of genomic DNA isolated from hatched larvae in the 750 ng pGL SMI treatment. SMI, described here for the first time, is the simplest and most efficient method for mass producing transgenic giant freshwater prawns. On the other hand, Microalga, Nannochloropsis oculata, is an important microorganism for feeding fish larvae. I develop transgenic lines of N. oculata that enable to produce an antimicrobial peptide, bovine lactoferricin (LFB), an algae-codon-optimized cDNA of LFB was fused with a red fluorescent protein (DsRed) reporter and driven by a heat-inducible promoter, which is a heat shock protein 70A promoter combined with a ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit 2’ promoter from Chlamydomonas reinhardtii. This construct was transferred into N. oculata cells by eletroporation. Two stable transgenic lines, which displayed a stable inheritance of transgene at least for 22 months, were obtained after I examined 491 microalgae clones. After the heat induction, the transcripts of LFB-DsRed gene were detected in the transgenic algal clone by RT-PCR. In addition, protein analysis showed that a band corresponding to the fusion protein of LFB-DsRed was positive in the immunoblotting analysis by using monoclonal anti-DsRed antibody. After I fed the transgenic algae by oral-in-tube to small model fish, medaka (Oryzius laptipes), I challenged by Vibrio parahaemolyticus infection for 6 h. The survival rate after 24 h infection of medaka fed with transgenic algae (1×108 cells/ per fish) was greatly higher than that of medaka fed with wild-type algae, 91.6% (n=12) versus 0% (n=6), suggesting that feeding the LFB- containing transgenic microalgae enables to protect medaka fish against V. parahaemolyticus infection.

並列關鍵字

Aquatic Strains ； Molecular Biotechnology

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