Heterogenous Expression Of Lutjanus Erythropterus And Lutjanus Fulviflamma Opsins

Red snapper(Lutjanus erythropterus) and Dory snapper(Lutjanus fulviflamma),belonging genus Lutjanus, which were warm water near the bottom of the case of offshore fish species of economic value for fishing higher species. Opsins play a crucial part in visual imaging and environmental adaptation. At present, for snapper fish at home and abroad research focused on the breeding technology and pathological research, the environment stress and system development. There is little research at the visual protein gene level. In this study, red snapper and Dory snapper as the research object, using genetic engineering to two snapper opsins long wavelength sensitive and Rhosopsin to build Prokaryotic expression vectorand eukaryotic expression. The main results are as follows:1.Using specific primers LWSF/R amplificat out the target gene LWS of red snapper,and than send to the Shanghai Sagon Company, insure the sequencing was correct, double enzyme, plastic recycling after that directed into the vector PET-32 a. After validation,make sure constructed a prokaryotic expression vector of red snapper gene LWS,and named pET-32a-LWS.2. Using specific primers ZLWSF/R amplificat out the target gene LWS of red snapper and Dory snapper, and than send to the Shanghai Sagon Company sequencing,insure it was correct, double enzyme, plastic recycling and then directed into the prokaryotic expression vector pPICZαA. After validation, make sure constructed two eukaryotic expression vectors of red snapper and Dory snapper gene LWS,and named as pPICZα-LWS-Ler and pPICZα-LWS-Lfu.3. Use the specific primers ZRHF/R condition PCR the target gene RH of red snapper and Dory snapper, and than send to the Shanghai Sagon Company sequencing, insure it was correct, double enzyme, plastic recycling and then directed into the prokaryotic expression vector pPICZαA. After validation, make sure constructed two eukaryotic expression vectors of red snapper and Dory snapper gene LWS, and named as pPICZα-RH-Ler and pPICZα-RH-Lfu.4.The eukaryotic expression vectors pPICZα-LWS-Ler,pPICZα-LWS-Lfu,pPICZα-RH-Ler and pPICZα-RH-Lfu linearizing by enzyme SacⅠ,were translated the vectors into Pichia pastoris by electric shock, inducible with methanol at 30 ℃,successfullyexpress the target protein red snapper LWS gene, Dory snapper LWS gene and Dory snapper RH gene, found the optimal abduction time was 12 hours.