Cloning, Expression Of The Hemocyanin Subunit TypeⅠGene From Locusta Migratoria Manilensis

Hemocyanins are copper-containing (Cu+) proteins that transport oxygen in manyarthropods hemolymph. It is colorless when deoxidized and blue when combined with oxygen.Insects possess an elaborate tracheal systemthat enables transport of gaseous oxygen from theatmosphere directly to the inner organs. Therefore, the presence of specializedoxygen-transport proteins in the circulatory system of insects has been considered generallyunnecessary. Only recently it has been demonstrated that hemocyanins occur in thehemolymph of many ametabolous and hemimetabolous insect taxa, but not in the Eumetabola(Hemiptera+Holometabola). Hexapod hemocyanins usually consist of two distinct subunittypes (Hc1and Hc2), Whereas type2subunits was absent in Orthoptera and Phasmatodea.Recent studies have shown that hemocyanin is a multifunctional protein. It not only withoxygen transport, but also with energy storage, the maintenance of osmotic pressure and theregulation of the molting process. Moreover, hemocyanin also have functions of phenoloxideactivity and anti-bacterial. It is considered to be an important arthropod immune molecules.The studies on cloning, expression, function of hemocyanins of Locusta migratoriamanilensis will bring momentous insights. It will help us to reveal the reaseans whyhemocyanin has been lost in some taxa and further understand the function of hemocyaninand its role in insect development. There are four Parts in the thesis:(1)The hemocyanin type1cDNA of L. m. manilensis(LmiHc1) was cloned using RT-PCR andRACE methods. The sequence was submitted to GenBank database(GenBank accession no.：HQ213937)and analyzed the nucleotides and deduced amino acids sequences. The full-lengthcDNA of Hc1gene is2271bp and the open reading frame is2016bp, which encodes a672amino acids protein with a calculated molecular mass of77.9KDa and the isoelectric point of6.06. N terminal signal peptide consists of18amino acids, LmiHc1mature peptide contains654amino acids. Sequence alignment analysis result showed that this gene shares94.7%identity with Schistocerca americana EHP.(2)Phylogeny trees reconstructed by Bayesian (BI) criteria using Hc1full-length sequencehighlights a consistent phylogeny of Orthoptera: the outgroup Collembola(S. curviseta)separated first from Insecta taxa at the base of the tree and insecta is monophyletic; in Insecta,M. germanica (Archaeognatha) and T. domestica (Zygentoma) were early separated being the most ancestral group which belong to Apterygota； in Pterygota, Blattaria was not amonophyletic group, it have close phylogenetic relationship with Mantcdea and Isoptera; thephylogenic relationships of these orders can be clearly denote as ((((((Blattaria, Mantcdea,Isoptera)(Orthoptera, Phasmatodea)) Plecotera) Dermaptera) Zygentoma) Archaeognatha).(3)With the aim of examining LmiHc1expression during the insect life cycle,semi-quantitative RT-PCR was performed using β-actin genes as internal control. A moderateamount of hemocyanin was found in all life cycle.(4)The LmiHc1prokaryotic expression vector was constructed by prokaryotic vectorpET-DsbA, and the recombined plasmid was transfected into BL21(DE3). After induction bycultivating37℃, eventually concentration IPTG1mM, the specific expression of theDsbA-fused protein was detected by SDS-PAGE. The result of SDS-PAGE showed that themolecular weight of the recombined protein is approximately60KDa, which is consistentwith its theory molecular weight. The Polypeptides of the Prokaryotie expression wereobtained.