文献类型： 会议论文

第一作者： Hong Wang

作者： Hong Wang ¹ ; Xiaohong Sun ² ; Aiping Feng ¹ ; Xiaoda Yang ³ ; Mingjie Chen ¹ ; Yingjie Pan ⁴ ;

作者机构： 1.Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Key Laboratory of Edible Fungi Resources and Utilization (South) , Ministry of Agriculture, China National Engineering Research Center of Edible Fungi, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai 201403, China

2.Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Key Laboratory of Edible Fungi Resources and Utilization (South) , Ministry of Agriculture, China National Engineering Research Center of Edible Fungi, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai 201403, China College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China Shanghai Ocean University, Shanghai 200090, China

3.Department of Chemical Biology, School of Pharmaceutical Science, Peking University, Beijing 100083, China

4.Shanghai Ocean University, Shanghai 200090, China

关键词： Volvariella volvacea;S-adenosyl-L-homocysteine Hydrolase (SAHH);S-Adenosyl-L-Homocysteine Hydrolase Gene (sahh);Real-time Quantitative PCR

会议名称： Congress of the International Society for Mushroom Science

主办单位：

页码： 240-246

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℃) gene expression determined using real-time quantitative PCR.

分类号： s646