Molecular Cloning And Prokaryotic Expression Of Growth Hormone Gene In Huso Dauricus

Huso dauricus originated 130 million years ago in the Cretaceous period. Natural populations are primarily distributed in the middle and lower reaches of the Heilongjiang River. H. dauricus is one of only two remaining Huso spp. in the world. H. dauricus is the largest freshwater fish in the world, and contains 3–5 times the quantities of unsaturated fatty acids and folic acid than those in any other fish, which makes them nutritious and delicious. The caviar, which is called “black gold”, is delicious but expensive. Therefore, H. dauricus has high economic value.Growth hormone(GH) is a single-chain polypeptide synthesized and secreted by the vertebrate pituitary gland that plays important roles in growth and development. GH promotes growth, accelerates protein synthesis, degrades lipids, and has other physiological functions in fish(Wei et al., 2004). It also improves feed conversion rate and regulates osmotic pressure in migratory fish. Therefore, GH has great practical value in aquaculture. However, the number of wild H. dauricus has plummeted, and individuals are smaller because of over-fishing. Moreover, this species has a long growth cycle; therefore, it is essential to promote research on the GH and related genes.The H. dauricus full-length GH c DNA was first cloned by real time-polymerase chain reaction(RT-PCR) and the rapid amplification of c DNA ends(RACE) techniques. We applied this sequence to further understand the molecular phylogenetics of H. dauricus, with the aim of researching GH functional characteristics and the evolution of the H. dauricus GH gene at the molecular level. We provide a theoretical basis to study the morphological classification H. dauricus and to apply the GH gene for practical production. We also quantified H. dauricus GH m RNA expression in different tissues to confirm that the GH gene contributes to growth, development, and reproduction. Finally, the prokaryotic expressionm vector was constructed to express the GH recombined protein, and the expression conditions were optimized, in order to lay a solid foundation for the development and use of H. dauricus GH gene.Firstly, RT-PCR was used to obtain the middle fragment of H. dauricus GH. Then, RACE was used to the obtain 3′, 5′-terminal fragment. The final spliced sequence was used to obtain the full-length H. dauricus GH c DNA, which was 1004 bp long, including a 5′ UTR of 47 bp, a 3′ UTR of 312 bp(30 bp Poly A is included), and an ORF of 645 bp. A BLAST analysis was conducted to determine the ORF base composition. As a result, GC content was 48.68%, AT content was 51.32%, and the gene was composed of 214 amino acids. The putative molecular weight and isoelectric point of the protein were 24.25 k D and 6.82, respectively. The translation initiation codon was ATG, and the termination codon was TAG. The gene sequence and the deduced protein sequence have been added to Gen Bank under accession number KP055783.The predicted H. dauricus mature GH amino acid sequence was very similar(up to 98.89%) to those of other Acipenseriformes. The homology analysis results showed 98.5% homology between the H. dauricus GH and that of H. huso, with disproportionation of 1.4%. H. dauricus was most unrelated to mammals, relatively unrelated to Ganoiidomorpha(Lepidosteiformes), and closely related to three species of Acipenseriformes.Secondly, We used Quantitative Real-time PCR to detect GH gene expression in muscle, gonad, stomach, the pituitary gland, and other H. dauricus tissues. The results show that GH m RNA expression was highest in the pituitary gland(P<0.05), followed by muscle, brain(except pituitary gland P<0.05). Very low expression was detected in the heart, stomach, gonad, intestine, spleen, liver and gill(P≥0.05).Thirdly, in order to construct recombinant plasmid Blunt E1-OFR, we took H. dauricus GH gene into the expression vector Blunt E1. The expression vector was transformed into the cells of BL21, and the expression conditions were optimized. After a lot of experiments found that, when IPTG was 0.6mmol/L, in the condition of 37 ℃cultured for 5h, could get a large amount of protein. The recombinant protein was confirmed by SDS-PAGE, and the molecular weight of the recombinant protein was 31 k Da, which was accord with the predicting outcomes.