Identification, Analysis and Expression of a Cold-induced Gene in Volvariella volvacea

摘要

Exposure of Volvariella volvacea, the Chinese straw mushroom, to low temperatures induces cell protein degradation and results in autolysis of the fungal mycelium. The full-length cDNA sequence of a coldinduced Cor3 gene from V. volvacea, identified by mRNA differential display technology, was digested with EcoR I restriction enzyme, ligated to the expression vector pPROK-1, and the recombinant plasmid was transformed into Escherichia coli JM109. The encoded protein was over-expressed in the transformed bacteria by addition of 0.5 mmol/L IPTG to the culture medium and cell-free extracts were prepared by sonication. DEAE-cellulose ion-exchange, Sephacryl S-200 gel filtration and QAE-Sepharose negative ion exchange chromatography yielded a single major protein band on SDS-PAGE with a molecular mass of 43 kDa and an isoelectric point of 6.1. The purified protein was identified as a S-adenosyl-Lhomocysteine hydrolase (SAHH) with a synthetic activity (measured by HPLC) of 4. 5 mol/(min ? mg) protein, and a hydrolytic activity (measured by titration of the product homocysteine with 5, 5'dithiobis (2-nitrobenzoic acid) of 0. 7 mol/(min ? mg). Plasmids containing parts of the sahh (target gene) and gpd (housekeeping gene encoding glyceraldehyde phosphate dehydrogenase) genes were constructed and low temperature (4°C) gene expression determined using real-time quantitative PCR.