Brachiostoma Belcheri Tsingtauense AmphiHSP90 Gene: Cloning, Tissue Expression And Immune Function Study

HSP90 family is a highly conserved family of proteins in evolutionarily, it has a high level in cells, as the case of heat shock, heavy metals, hypoxia, acidosis, infection of bacteria or virusessuch, it may be doubled its content. More and more evidences showed that, HSP90 could be involved in innate and acquired immune responses.Amphioxus is a transition type located between invertebrates and vertebrates, and is the valuable material in the study of vertebrate evolution. In this paper, the HSP90 gene of amphioxus was cloned by PCR. The primary structure of the putitive protein fragment of AmphiHSP90 was predicted. Homology analysis of AmphiHSP was completed. The expression level of RNA or protein of AmphiHSP90 in normal adult amphioxus tissues was detected by ISH or IMMS. The difference changes of this gene in expression under the conditions of the impact of pathogenic micro-organisms in adult amphioxus tissues compared with that of health amphioxus'was showed by PCR. Those resultes provided a series of experimental evidences for studying its structure, the evolution and the role in immune process of amphioxus.The cloned AmphiHSP90 fragment is 1481bp in length, encoded the protein fragment composed of 493 amino acids. The AmphiHSP90 protein has a typical conserved HSP90 Domain and has high identities (among 80%-85%) with the HSP90 proteins of other species, such as Pan troglodytes, Rattus, mice, Gallus gallus, Xenopus, Zebrafish as well as Drosophila.Phylogenetic analysis showed that the AmphiHSP90 protein position is between vertebrates HSP90 protein and invertebrates HSP90 protein, and the evolution of the gene is basically the same as the phylogenetic position of amphioxus.In situ hybridization results of adult amphioxus tissues showed that, AmphiHSP90 mRNA is widely expressed in amphioxus tissues (including skin, gill filaments, intestinal epithelium, Gill filaments, Intestinal, testis membrane and ovary membrane). But the immunohistochemistry results showed that AmphiHSP90 protein expression is a large number of columns in the expression of muscle and gill filaments, a small amount of expression in the epidermis in amphioxus body. The Inconsistency between AmphiHSP90 protein expression pattern and the gene expression patterns might be caused by two reasons:One reason is that amphiHSP90 protein is very much rich in, muscle of adult amphioxus alreadly, since that HSP may be exercised the role of molecular chaperone in the muscle cells, and can be binded to muscle actin, microtubule binding protein and tyrosine kinase involved in essential metabolic activities of cell; On the otherwise, the mRNA rich expression of HSP in Various epithelial tissues maybe related to the secretion of HSP90. The other reason is that the credibility of experimental results obtained by heterologous antibody may be relatively low.So the protein expression should be deeply identified in further studies.Some health adult amphioxus were immune- impacted by using E.coli or S.aureus gave, then the level of AmphiHSP90 gene expression were measured with semi-quantitative RT-PCR. The resultes detected that no matter impacted by E. coli or S. Aureus, AmphiHSP90 expression did not change significantly in control group; AmphiHSP90 expression were significantly increased after impact in experimental group, suggesting that HSP90 might play a protective role in resistingance to the bacteria infection outside world for amphioxus.