Transgenic Tiger Shrimp (Penaeus monodon) by Electroporation with Reporter and Porcine Lactoferrin Gene

• Main Authors: Fu-Shen Tseng, 曾福生
• Other Authors: Huai-Jen Tsai
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/61923217248310647543

博士 === 國立臺灣大學 === 動物學研究研究所 === 92 === In the past 30 years, the shrimp-culture industry has grown rapidly and so has the number of shrimp pathogens. To date, at least 20 virus to infect shrimp, only six viruses were known to infect shrimp in 1988, but that number climbed to 11 in 1993, 14 in 1996, and more than 20 in 1998. White spot syndrome virus (WSSV) is one of the most important viral pathogens infecting commercially cultivated shrimp species around the world. Because WSSV can cause up to 100 % mortality, its effect on the shrimp-culture industry and can be devastating. WSSV can be detected easily with a PCR diagnostic kit, but there is no effective treatment. Recently, a number of anti-microbial and immuno-modulation genes have been cloned and proven to confer resistance to bacteria and viral pathogens. Lactoferrin (LF), glycosylated protein produced by lactating mammary glands and the epididymis. LF has a net positive charge and an iron-binding glycoprotein able to transport metals, and has bacteriostatic and bactericidal activity against Gram-negative and Gram-positive bacteria. is part of the first line of immune system defense against bacteria, fungi and viruses. The disease resistance of anti-microbial function might offer the new view for the protection from pathogen in shrimp culture. Furthermore, to study transgenic tiger shrimp Penaeus monodon introducing reporter gene (pFLAG-CMV-1-BAP) into zygotes of tiger shrimp by electroporation. Set up the conditions of electroporation. We constructed an expression vector pCMVLF, which contains porcine LF gene, produced transgenic tiger shrimp to study resistance for WSSV-infection, and the resistance was transmitted from parental transgenic tiger shrimp to F1 progeny. A reporter gene driven by the promoter regulatory region of the human cytomegalovirus (CMV) fusion baterial alkaline phosphatase foreign gene (pFLAG-CMV-1-BAP) into penaeus monodon fertilized egg. The transgenic tiger shrimp was achieved by using 10 kv; 28 pulses; 120 μsec pulse time; 10 cycles; and a concentration 37.5 μg/mL. A dot blot analysis, the rate of gene transfer was 37 % in mysis-stage, 23 % postlarva 15 (PL15), 19 % postlarva 45 (PL45), and 21 % 4-month-old (about PL120). Analysis of genome of transgenic tiger shrimp by Southern blotting revealed that pFLAG-CMV-1-BAP were 31 % integrated into the genomes of the transgenic tiger shrimp and mosaically distributed in a wide variety of tissues. Immunohistochemical staining revealed that the FLAG-BAP fused-protein encoded by pFLAG-CMV-1-BAP was present in the ovaries of some transgenic tiger shrimp. Therefore, we constructed a plasmid that included a porcine lactoferrin (pLF) gene driven by CMV (pCMVLF). After 22 months, the average growth was larger and the mortality of the transferred shrimps was lower, compared to these of mock-treated and untreated shrimps. Southern blotting analysis of genomic DNA from 10-month-old transferred shrimps determined that 4.9 % were transgenic, and 25 % transgenic shrimp expressed pLF. To study resistance of P1 transgenic shrimp expressing pLF to WSSV, which were reared with WSSV-infected farm shrimp. After 2 months, the survival rate was 81�b20 % more than the untreated 12�b10 % and mock-treated 18�b2 %. Porcine LF levels in the hemoplasma ranged from 0.84 to 2.28 μg/mL, this transgenic shrimp had significantly higher fiber content than other shrimp. There were 19 % of F1 progeny bearing pLF gene, produced by P1 male transgenic shrimp crossed with wild female. Western blotting analysis F1 progeny showed that there were 14.3 % expression pLF. To challenge F1 progeny with white spot syndrome virus (WSSV), the F1 progeny were reared with WSSV-infected farm shrimp. After 21 days, the survival rate was 47 % significantly more than the control. This suggested that transgenic shrimp expressing pLF are resistant to WSSV infection and transmitted.