Cloning And Expression Analysis Of Hsp90Gene From The Chinese Oak Silkworm,Antheraea Pernyi

Antheraea pernyi is a kind of deveoping wild ilk-producing insect, which blongs to Lepidoptera Saturniidae Antheraea. Antheraea pernyi has good officinal and edible economical values except for utilizing in spinning industry as raw materials. Oak cocoon to make use of raw material of textile industrial, and the pupa also have high medicinal value and edible value. Up-regulation of Hsp70and Hsp90mRNA levels in response to cold and heat hardening, density, starvation, diapause and developmental stages were reported in several insects. However, there is no published information about the stress-induced HSPs gene sequences and their expression from A. pernyi. The heat shock proteins (HSPs). which were found in most organisms are highly conserved in all eukaryotes and prokaryotes so far studied. HSPs appear to be also involved in various biological processes including cell proliferation and differentiation, embryogenesis and aging. This study is to examine the thermal responses among different developmental larvae and pupae of the species on the basis of cloning, sequencing and expression of Hsp90. The results are as follows:1. cloning and sequence analysis of HSP90cDNAA full-length cDNA encoding Hsp90from A. pernyi was cloned and characterized by RT-PCR and RACE technique. The complete cDNAs (2482bp) contains a2154bp open reading frame encoding717amino acid residues and with a predicted molecular weight of82.6kDa and an isoelectric point of5.0. The nucleotide sequence of the cDNA is highly similar with the Hsp90cDNAs of some other insect species, and it includes an important and intact Hsp90signature sequence. Phylogenetic analysis indicated that A. pernyi Hsp90gene had the maximum homology with A. yamamai Hsp90.2. The expression of mRNA levels of Hsp70and Hsp90in larvae stressed by high temperaturesThe total RNA of A. pernyi was extracted to analyze the expression of Hsp90in different tissues, then reversely transcribed and synthesized the frist cDNA by M-MLY. The result showed that Hsp90of A. pernyi ubiquitously expressed in all examined tissues. There is no significant difference between4tissues of gene expression except for testis.A real-time fluorescence quantitative RT-PCR was constructed to examine the transcriptional expression levels of Hsp90from oak pupa of A. pernyi under t high temperature stress (42℃) for30min. respectively, and corrected the result with18S rRNA gene of A pernyi. The result showed that high temperature could induce the expression of Hsp90obviously. These results suggested that Hsp90play an important role in adaptation to hot temperature stress which can increase their tolerance to extracellular heat shock.3. Prokaryotic expression of Ap-hsp90Hsp90gene was amplified by PCR, ligated to pET-28a(+) expression vector and transformed into Escherichia coli BL21(DE3), induced by IPTG under different concentrations. Through SDS-PAGE and purification analysis, the induced fusion protein was successfully expressed.