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首页> 外文会议>Proceedings of the 18th congress of the International Society for Mushroom Science >Identification, Analysis and Expression of a Cold-induced Gene in Volvariella volvacea
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Identification, Analysis and Expression of a Cold-induced Gene in Volvariella volvacea

机译：草菇中感冒基因的鉴定，分析与表达

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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.
机译：禾本科植物草菇Volvolvella volvacea暴露于低温下会诱导细胞蛋白降解，并导致真菌菌丝体自溶。用mRNA差异显示技术鉴定的冷链霉菌Cor3基因全长cDNA序列，用EcoRI限制性酶消化，与表达载体pPROK-1连接，并将重组质粒转化到大肠杆菌JM109中。通过向培养基中添加0.5 mmol / L IPTG，在转化的细菌中过表达编码的蛋白质，并通过超声处理制备无细胞提取物。 DEAE-纤维素离子交换，Sephacryl S-200凝胶过滤和QAE-Sepharose负离子交换色谱在SDS-PAGE上产生单个主要蛋白带，分子量为43 kDa，等电点为6.1。纯化的蛋白质被鉴定为具有4. 5 mol /（min？mg）蛋白质的合成活性（通过HPLC测量）和水解活性（通过滴定高半胱氨酸产物测量）的S-腺苷-Lhomocysteine水解酶（SAHH）。用0. 7 mol /（min？mg）的5，5'二硫代双（2-硝基苯甲酸）构建含有sahh（靶基因）和gpd（编码甘油醛磷酸脱氢酶的管家基因）部分基因的质粒，并进行低温培养实时定量PCR确定（4℃）基因表达。

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  • 会议地点 Beijing(CN)
  • 作者

    Hong Wang; Xiaohong Sun; Aiping Feng; Xiaoda Yang; Mingjie Chen; Yingjie Pan;

  • 作者单位

    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;

    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;

    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;

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

    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;

    Shanghai Ocean University, Shanghai 200090, China;

  • 会议组织
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 菌类（食用菌）;
  • 关键词

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

    机译：草菇; S-腺苷-L-高半胱氨酸水解酶（SAHH）; S-腺苷-L-高半胱氨酸水解酶基因（sahh）;实时定量PCR;

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