Improve Aquatic Strains by Using Molecular Biotechnology

• Main Authors: Si-Shen Li, 李思賢
• Other Authors: Huai-Jen Tsai
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/31741859910644018328

博士 === 臺灣大學 === 分子與細胞生物學研究所 === 96 === 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.