Molecular Cloning Of Growth Hormone CDNAs From Pleuronectiformes Fishes And Study On Saccharomyces Cerevisiae Expressing Flounder Growth Hormone

In this paper,Growth hormone cDNAs of 9 Pleuronectiformes fishes were cloned and the phylogenetic analysis via these sequences was performed by PAUP software.Four vectors expressing flounder GH in Saccharomyces cerevisiae were constructed and studies on the transgenetic yeast were made.The mRNA of pituitary glands from seven important economic Pleuronectiformes fishes was extracted,the cDNA sequences of GH(Growth Hormone) gene were then isolated from Cleisthenes herzensteini,Limanda yokohamae,Pleuronichthys Cornutus,Cynoglossus robustus,Platichthys bicoloratus, Zebrias zebra and Paralichthys dentatus with RT-PCR method by using designed specific primers based on the reported GH cDNA sequences of Pleuronectiformes. The results of sequencing showed that lengths of the above cDNAs are as follows: 479 bp,564 bp,519 bp,440 bp,564 bp,440 bp and 522 bp.The GH cDNA sequences encode mature polypeptide of about 140 to 170 amino acid.The full-length growth hormone cDNAs of Paralichthys lethostigma and Scophthalmus maximus were cloned by Switching Mechanism At 5' end of the RNA Transcript (SMART) RACE technology.The GH full-length cDNA of Scophthalmus maximus is 876 nucleotides long,codes for a polypeptide of 197 amino acids and the GH full-length cDNA of Paralichthys lethostigma is 902 nucleotides long,codes for a polypeptide of 190 amino acids.The deduced GH amino acid sequences of 15 Pleuronectiformes and 7 outgroup species were used for the phylogenetic analysis, which was performed by PAUP software with the maximum parsimony method.The result was consistent with morphology of Pleuronectiformes on the whole.In the topology founded,Scophthalmus maximus and Solea senegalensis formed an independent branch each other and showed far relationship with species of Bothidae and Pleuronectidae,the phylogenic position of Solea senegalensis is accorded with the reported result of analysis made by mitochondrial genome.It seems reasonable to speculate that GHs can be effectively used in analyzing genetic relationship and taxonomic status of Pleuronectiformes and other species of teleostean.The non-integrated vector pYD-GH was constructed by inserting fGH gene into yeast surface display vector pYD1.And three integration vector,E2 used for expressing on cell surface by induce,E4 used for expressing in cell by induce and EP4 used for expressing in cell not by induce.The Vectors were transformed into Saccharomyces cerevisiae strain EBY100 to expressing flounder growth hormone, then expression and biological activity of heterologous protein were studied.The non-integrated vector pYD-GH in yeast cells can not be passaged in the presence of stable,but the positive rate of integration vector pYD-E2,pYD-E4 and pYD-EP4 after 30 generations was 100%,which means fGH gene had been integrated into the yeast genome through homologous recombination.The fluorescence microscopy and flow cytometry analysis of fluorescent marked pYD-GH and pYD-E2 showed that the yeast has the highest intensity of the fluorescence after galactose induced 6 hours and after induced 12 hours the yeast still has higher fluorescence intensity.After calculation,the expression amount of yeast strain E2 with GAL promoter for extracellular expression of flounder growth hormone was 1.02‰,the expression amount of E4 with GAL promoter for intracellular expression was 1.49‰,and the expression amount of EP4 with PGK promoter for intracellular expression was 1.93‰.The detection of biological activity showed that E2,E4 and EP4 were all in prominent role of promoting growth of flounder and conversion rate of food.Even if the addition was only 0.5%,weight of flounder increased 27.16%,26.76%and 31.21%more than the control group in the seven weeks study,conversion rate of food also increased 22.75%,22.34%and 26.15 more than the control group.When addition of Saccharomyces cerevisiae was up to 1%,the weight of fish feed with transgenic Saccharomyces cerevisiae(E2,E4,EP4) increased 47.29%,46.87%and 50.24% more than the control group,conversion rate of food also increased 30.65%,30.46% and 32.07%more than the control group. Finally,acute toxicity test,the traditional teratogenic test,bone marrow and sperm abnormality micronucleus test were performed on KM mice by the oral route for evaluating safety of transgenic Saccharomyces cerevisiae.Acute toxicity test results showed that transgenic Saccharomyces cerevisiae had no toxic to mice (LD50＞10g/kg).In the traditional teratogenic test,bone marrow and sperm abnormality micronucleus test and experiments of impact in mice organs,three yeast strains of the three-dose(2.0 g/kg,5.0g/kg and 10.0 g/kg) compared to the control group were not significantly different(P＞0.05).These results suggest that Saccharomyces cerevisiae transformed with flounder growth hormone does not produce teratogenic and mutagenic effects in mice,and may be a safe transgenic feed additives.