Cloning And Heterologous Expression Of Heat Shock HmHSP70 Gene From Hypsizygus Marmoreus

Hypsizygus marmoreus, a rare edible fungus, is popular by its delicious taste and nutritious qualities. H. marmoreus belongs to low temperature type of edible fungi which production was impact with higher(>25℃) or lower(<5℃) temperature. Heat shock proteins are the most important adversity induced proteins in organism. They are speedy synthesized and protected cell avoid damage when organism suffering adversity especially temperature stress. Depend of their molecular chaperone function they participated in various cellular processes under normal and stress conditions. They are also playing key role in the processes of organism defend environment stress such as heat, cold and oxidation. Accordingly, the heat shock genes are fine genes resources which have important practical valuable in breeding work. Heat shock protein has been studied mainly in animals and plants at home and abroad, whereas rarely in fungi. The heat shock protein from mushroom hasn't been reported yet.The total length of HmHSP70 cDNA sequence was cloned successfully by RACE from H. marmoreus in this study. Recombinant HmHSP70 was overexpressed in Escherichia coli. Partial function of the protein was tested in this study. The results were as follows:1,The degenerate primers were designed according to conserved sequences of heat shock gene from Achlya klebsiana (AKU02504), Candida glabrata (AY077689), Rhizopus stolonifer (AY147869) in GeneBank with Primer Premier5.0 and DNAMAN6.0. The partial fragment of HmHSP70 gene was amplified with degenerate primers and was constructed with pMD18-T vector which was verified by PCR amplification. The recombinant plasmid was transferred to E.coli competent cell and sequenced by Shanghai Sangon biological engineering technology and service Co., Ltd. The fragment contains 690 nucleotides coding 230 amino acid redidues.2,The specific primers were designed by result of DNA sequencing. The total RNA was extracted by Trizol method from H. marmoreus mycelium which was heated by 42℃. The 3'ends and 5'end of the gene were cloned by PT-PCR and RACE methods and constructed with T vector. The new primers were designed according to the results of sequencing by Shanghai Sangon biological engineering technology and service Co., Ltd. The HmHSP70 gene contains 2308 nucleotides with a 2001 nucleotides length open reading frame (ORF) coding 667 amino acid residues (GenBank access number is GQ246176).3,Recombinant vector pET32a-c(+)/HmHSP70 was overexpressed in E.coli BL21(DE3). The result of SDS-PAGE was indicated that the predicted molecular weight of recombinant protein was 92KDa that coincided with our expectation by IPTG induced. By the heat shock experiments, the results of cell viability and SDS-PAGE analysis of cell lysates suggested that these recombinants could improved the capacity of E.coli to against higher temperature (50℃) and could protected other proteins in cells.4,DNA sequence and the amino acid sequence of HmHSP70 in H. marmoreus were analyzed by the methods of Bioinformation. This gene belongs to HSP70 gene family. The phylogenetic tree is formed based on the sequence alignment which showed the similarity between H. marmoreus and Laccaria bicolor (XP₀01880175), H. marmoreus and Cuyitococcus neofonmans (XP₅67978), H. marmoreus and Aspergillua clavatus (XP₀01275300), H. marmoreus and Schizosaccharomyces (XP₀02172864) was 87%, 71%, 67%, 67%, respectively. The molecular weight of HmHSP70 is 72.2KDa, PI is 5.63, the instability coefficient is 36.67. The hydrophobic property analysis indicated that it was soluble protein.The results of this experiment establish a favorable foundation for varieties improvement in H. marmoreus, the research on heat shock protein and molecular breeding in fungi.