Molecular cloning, antiserum preparation and expression analysis during head regeneration of α-crystallin type heat shock protein in Hydra vulgaris

Journal of genetics

WEN-FANG DONG^1¹ 

[1] Provincial Key Laboratory of Conservation and Exploitation of Biological Resources in Anhui, Provincial Key Laboratory of Biotic Environment and Ecological Safety in Anhui, College of Life Sciences, Anhui Normal University, Wuhu 241000, Anhui Province, People’s Republic of China^1

关键词: α-crystallin type heat shock protein;    polyclonal antiserum;    quantitative real-time PCR;    whole-mount immunohistochemical assay;    RNA interference;    Hydra vulgaris.;   

DOI  :  

学科分类：生物科学（综合）

来源: Indian Academy of Sciences

【 摘 要 】

Our previous study based on the transcriptome profiling indicated that a fragment of α-crystallin type heat shock protein (α-Hsp) gene was one of the numerous cDNA sequences expressed differentially at various stages of head regeneration in Hydra vulgaris. To further investigate the role that which α-Hsp plays during hydra regeneration, a full-length cDNA of α-Hsp gene of H. vulgaris was isolated by the rapid amplification of cDNA ends (RACE) technique. The full-length cDNA of α-Hsp gene was 1156 bp, containing a 765 bp open-reading frame (ORF), which encodes a polypeptide of 254 amino acid residues with a molecular weight of 29.27 kDa. Further, the ORF was subcloned into the plasmid pET-42a(+), and the recombinant plasmid pET-42a(+)-α-Hsp was transformed to Escherichia coli BL21(DE3), then the fusion protein GST–α-Hsp was expressed mainly in the form of a soluble molecule after induction by isopropyl-β-d-thiogalactopyranoside. In addition, BALB/Cmice were immunized with the fusionprotein to prepare the polyclonal antiserum which was used as the primary antibody for whole-mount immunohistochemical assay. The results from the immunohistochemical assay showed that α-Hsp had expressedmainly at the wound site and nearby area of hydra after decapitation operation, and both quantitative real-time polymerase chain reaction (qPCR) analysis and immunohistochemical assay revealed that the expression level of α-Hsp increased gradually during the early period of hydra regeneration, then reached apeak at 24 h after decapitation operation, while decreased during the late regeneration period. Moreover, it indicated an important role of α-Hsp gene in hydra head regeneration that RNA interference (RNAi)-mediated α-Hsp silencing led to the obvious delay of the regeneration of head structures in H. vulgaris. In conclusion, our results gave the hint that α-Hsp could be related to wound healing and tissue remodelling at early regeneration stages, and may lay the foundation for further studies about the physiological function and role of α-Hsp during hydra regeneration.

【 授权许可】

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