Cloning And Expression Of The Heat Shock Protein 70 Gene From Mantichorula Semenowi

Mantichorula semenowi Reitter,1889 (Coleoptera:Tenebrionidae) is an endemic species distributed in the floating dune of Southeast Alxa, China and South Mongolia which adjoins China. The species evolved special physiological and ecological characteristics of adaptation under environmental stress. Its imago can survive on the surface of dune with extreme temperature as high as 70℃and use the microhabitat to produce offspring. The present results provide new insights into mechanisms used by M. semenowi to adapt to stressful environments.This thesis contains 3 parts.1.The cloning and expression analysis of Mshsp70 and Mshsc70 from M. semenowi. The cDNA was cloned using RT-PCR and RACE methods.2.The cloning and expression analysis ofβ-actin from M. semenowi. The cDNA was cloned using RT-PCR and RACE methods, and detected reliability ofβ-actin genes as internal control within insects after heat shock. To establish a stable expression in vivo geneticβ-actin steward for the internal control semi-quantitative RT-PCR method. By this method of heat shock the gene mRNA expression level of Mshsp70 and Mshsc70 relative quantitative were researched in M. semenowi.3.The Mshsp70 prokaryotic expression vector was constructed and in DE3 induction expression, to purify the protein making antibody in rabbit.The results are offered as follows:(1) The full-length cDNA of Mshsp70 is 2137 bp, containing a 3'-untraslate region of 168 bp, which encords a deduced 649 amino acid peptide with a predicted molecular mass of 71.34 KD, the result of sequence alignments analysis showed that this amino acids shares 80% identity with Harmonia axyridis and protein shares 85% identity with Tribolium castaneum.(2) The cDNA of Mshsc70 is 1908 bp, containing a 3'-untraslate region of 100 bp, which encords a deduced 601 amino acid peptide with a predicted molecular mass of 57.85 KD, the result of sequence alignments analysis showed that this nucleotide and protein shares 80% and 85% identity with Tribolium castaneum respectively.(3) The full-length cDNA ofβ-actin is 1372 bp, containing a 5'UTR of 66 bp and 3'UTR of 175 bp. The open reading frame ofβ-actin is 1131 bp, which encords a deduced 376 amino acid peptide. The result of sequence alignments analysis showed that this nucleotide share 96-99% identity with other animals. The sequence of theβ-actin was submitted to GenBank and assigned the accession number EU825991. Profiles ofβ-actin expression in different recovering time after heat shock were expressed similarly and not significantly different with that of the unstimulated control, suggesting an ubiguitous expression pattern can be used as internal control in gene mRNA expression level.(4) Semi-quantitative RT-PCR analysis showed that 1 h heat shock treatment at 42℃caused rapid increase of Mshsp70 mRNA, which reached maximum levels at the end of the heat shock treatment, and decreased gradually after being moved to room temperature. Expression of Mshsc70, however, was inhibited after heat-shock treatment:during recovering 2-4 h, the expression levels were only little times that of the control.(5) The Mshsp70 prokaryotic expression vector was constructed by prokaryotic vector pET-DsbA, and the recombined plasmid was transfected into DE3. After induction by cultivating 37℃, eventually concentration IPTG 1mM, the specific expression of the DsbA-fused protein was detected by SDS-PAGE. The result of SDS-PAGE showed that the molecular weight of the recombined protein is approximately 59.4 KD, which is consistent with its theory molecular weight. Using His affinity chromatography purified get fusion protein, to preparation HSP70 antibody, after western blot detected and expected the same size of specific bands.