| The remarkable tolerance of Ericerus pela to extreme hot and cold temperatures is important for its population distribution and wax production. The correlation between heat shock protein (Hsp) and tolerance of Ericerus pela to high/low temperatures and the correlation between Hsp and Ericerus pela developmental process were investigated. Quantitative real time PCR (qRT-PCR) and Rapid amplification of cDNA ends (RACE) were used. Based on7hsps (hsp21.5, hsp21.7, hsp40, hsp60, hsp70, hsc70and hsp90) sequences selected from the E. pela transcriptome, transcriptional expression pattern of the7hsps during heat/cold stress and development was examined. For temperature-stress tolerance analysis, larvae and adults were selected as two representative developmental stages. Furthermore, the full length gene sequences of hsp20family members (hsp21.5and hsp21.7) and hsp70family members (hsp70and hsc70), which were significantly changed in respond to heat/cold shock and during development, were cloned and characterized. The results showed that:(1) hsp21.7, hsp60, hsp70, hsp90were sensitive to heat and cold stimulus:heat stress:①Adult females. All the seven hsps were responsive to heat stress. The expression of hsp21.7and hsp70were drastically up-regulated; hsp90mRNA expression level was also increased.②Male second-instar larvae. hsp21.7and hsp90were induced significantly; however, there was down-regulated mRNA expression of hsp21.5and hsc70.cold stress:In adult females, all the ep-hsps were induced by cold stress except hsp40. hsp21.5and hsp70were strongly up-regulated.(2) hsp21.5, hsp40, hsp60, hsp70, hsc70had correlation with development regulation:①In females, all the7hsps were up-regulated during male development stages except hsp21.7and hsp90. The expression of hsp21.5reached a maximum in second instar larvae; hsp40, hsp60and hsp70were up-regulated significantly in adult; hsc70, in the first instar larvae.②n males, all the hsps were up-regulated. The mRNA level of hsp21.5was maximal in adults; the mRNA levels of other hsps peaked in second instar larvae.(3) The sequence features of ep-hsp20The full-length sequences of ep-hsp21.5and ep-hsp21.7were997and873bp and contained570and564bp opening reading frames (ORFs), respectively. The ORP of ep-hsp21.5encoded a putative protein of189amino acids with a calculated molecular mass of21.5kDa, and the ORF of ep-hsp21.7encoded a deduced protein of187amino acids with a molecular mass of21.7kDa. ep-hsp21.5and ep-hsp21.7both contained the conserved a-crystallin domain, indicating that the isolated ep-hsp21.5and ep-hsp21.7sequences were small Hsp genes (shsp). The phylogenetic tree analysis showed that ep-Hsp21.7was clustered into the species-specific sHsp group, while ep-Hsp21.5was clustered into the orthologous sHsps group.(4) The sequence features of ep-hsp70Full-length cDNA sequences of ep-hsp70and ep-hsc70were2,355and2,417bp and contained1,908and1,962bp ORFs, respectively. ep-hsp70encoded a deduced protein of635amino acids with a calculated molecular mass of69.8kDa. ep-hsc70encoded a deduced protein of653amino acids with a calculated molecular mass of71.6kDa. The three characteristic signature sequences of the Hsp70family were observed in the amino acids sequences of ep-Hsp70and ep-Hsc70. The phylogenetic tree analysis of Hsp70showed that ep-Hsp70was clustered together with inducible Hsp70, whereas ep-Hsc70was clustered with cognate Hsc70. Both ep-Hsp70and ep-Hsc70were clustered with cytoplasmic Hsp70s. The genomic DNA sequence of hsc70had four introns, which were1139,255,514and94bp in length. The first intron was located in the5’UTR, while the remaining three introns were located in the coding regions.In E. pela, inducible hsps, such as ep-hsp21.7and ep-hsp70, were involved in temprature stress; whereas ep-hsp21.5and ep-hsc70were constitutive, and were involved in development regulation. These results were similar to most researches. While in extreme temprature, constitutive hsp could also paly a role in temprature stress. |
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