Study On The Growth-promoting Effect On Fish Of Transgenic Synechocystis PCC 6803 Containing Flounder Growth Hormone Gene And Its Safety Evaluation

Fish growth hormone (GH), a promoter of fish growth, should be of great value in fish culture, and it has already gained great attention. The exploiture of fish GH for its application in aquaculture are mainly through the breeding of GH transgenic fish and the expression of GH gene in gene engineering host. Because of the food safety and environmental safety issues accompanied with transgenic fish, the use of recombinant growth hormone (rGH) in aquaculture has generated considerable interest. In this paper, the growth-promoting effect of transgenic Synechocystis sp. PCC6803 containing flounder GH gene constructed by our lab is studied, and the safety of this transgenic alga on mouse, flounder, turbot, hepatocytes of flounder and tonguefishin in primary culture are evaluated. The present study provides theory and experience for the use of the transgenic alga in fish culture production.Studies on growth-promoting effect of transgenic Synechocystis include three parts: the first is growth-promoting effect on flounder, the second is growth-promoting effect on turbot and the third is growth-promoting effect on hepatocytes of flounder and tonguefishin. 7-week flounder feeding experiment and 40-day turbot feeding trial show that transgenic Synechocystis sp. PCC6803 containing flounder GH gene can significantly enhance fish growth and improve feed conversion ratio. 0.5% and 2.0% dose of transgenic alga increase body weight of flounder by 32.09 % and 49.04 % than that of control group, respectively. Enhancement in feed conversion ratio of flounder in 0.5% and 2.0% transgenic alga groups are 20.75% and 35.85%, respectively. In turbot, 1.0% transgenic alga causes 18.54% and 9.84% increase in body weight and feed conversion ratio (P < 0.05), respectively. Neutral red uptake and MTS reduction assays verified that hepatocytes of flounder and tonguefishin in primary culture exposed to 100μg/ml and 200μg/ml of transgenic Synechocystis significantly elevated relative survival rate (P < 0.05).To evaluate the safety of transgenic Synechocystis containing flounder GH gene, acute toxicity, traditional abnormality induction, bone marrow erythrocyte micronucleus and sperm abnormality test in mice; survival rate, muscle composition, blood physiological and biochemical parameters, peripheral blood erythrocyte micronucleus and other nuclear abnormality, organ/weight ratio and histopathology examination in fish; LDH release and DNA fragmentation assay in hepatocytes of flounder and tonguefishin are conducted. Mice toxicity assays show that the LD50 of transgenic Synechocystis is more than 10g/kg; reproductive function of female mice and body weight and length of fetal mice are not affected by transgenic alga; neither abnormality of skeleton, organ and sperm nor micronucleus of bone marrow erythrocyte is induced by the transgenic alga. Test on organ/weight ratio of heart, liver, spleen, and the histopathology examination on stomach, intestine, pyloric caecum, liver, spleen and kidney in flounder and turbot verify that transgenic alga can not damage fish organ structure. Analyses on haematological parameters reveal that the transgenic alga has no effect on fish blood cell count, haemoglobin level, serum aminotransferase activities, and metabolite content, such as protein, glucose, triglyceride, cholesterol, creatinine and so on. Frequencies of micronucleus and other nuclear abnormality in erythrocytes of turbot show no difference among transgenic alga groups and control. Muscle composition analyses show that feeding transgenic alga can not influence proximate composition of muscle in flounder, but significant (P<0.05) decrease in moisture content and significant (P<0.05) increase in protein content (12.73%) is induced by transgenic Synechocystis. However, lipid and amino acid profile is not changed by transgenic alga. Examination on the morphology, LDH release and DNA fragmentation of hepatocytes in flounder and tonguefishin demonstrate that the cell exposed to transgenic alga showed similar structure and LDH release to the control. No DNA fragment is detected in cell treated with transgenic Synechocystis.Muscle of flounder fed with transgenic Synechocystis is fed to mice by gavage to study if the transgenic alga can influence animal fed with the fish through food chain. Results illustrate that the mice fed with experimental fish are not significantly different from the control mice in growth, blood cell count, haemoglobin content, serum aminotransferase activities, histological structure and reproductive function.Results of present study indicate that transgenic Synechocystis sp. PCC6803 containing flounder GH gene is an efficient growth promoter and a safe feed additive for fish. It could bring great benefits to fish farmers.