編碼S-腺苷-L-高半胱氨酸水解酶的草菇冷誘導(dǎo)基因Cor3功能驗(yàn)證
Cold-Induced Volvariella volvacea Cor3 Gene Encodes an S-Adenosyl-L-homocysteine Hydrolase
【作者】 孫曉紅； 汪虹； 楊曉達(dá)； 馮愛(ài)萍； 陳明杰； 潘迎捷；
【Author】 SUN Xiaohong1,2,3,WANG Hong1,YANG Xiaoda4,FENG Aiping1,CHEN Mingjie1,PAN Yingjie3(1Institute of Edible Fungi,Shanghai Academy of Agricultural Sciences;Key Laboratory of Edible Fungi Resources and Utilization(South),Ministry of Agriculture,P.R.China;National Engineering Research Center of Edible Fungi;National R&D Center for Edible Fungi Processing;Key Laboratory of Agricultural Genetics and Breeding of Shanghai,Shanghai 201403,China;2College of Life Sciences,Nanjing Agricultural University,Nanjing,Jiangsu 210095,China,3Shanghai Ocean University,Shanghai 201306,China; 4Department of Chemical Biology,School of Pharmaceutical Science,Peking University,Beijing 100083)
【機(jī)構(gòu)】 上海市農(nóng)業(yè)科學(xué)院食用菌研究所,農(nóng)業(yè)部南方食用菌資源利用重點(diǎn)實(shí)驗(yàn)室,國(guó)家食用菌工程技術(shù)研究中心,國(guó)家食用菌加工技術(shù)研發(fā)分中心,上海市農(nóng)業(yè)遺傳育種重點(diǎn)開(kāi)放實(shí)驗(yàn)室； 南京農(nóng)業(yè)大學(xué)生命技術(shù)學(xué)院； 上海海洋大學(xué)； 北京大學(xué)醫(yī)學(xué)部藥學(xué)院；
【摘要】 將草菇(Volvariella volvacea)冷誘導(dǎo)基因Cor3cDNA序列經(jīng)EcoR I酶切克隆到原核表達(dá)載體pPROK-1中,轉(zhuǎn)化大腸桿菌(Escherichia coli)JM109,經(jīng)異丙基-β-D-硫代吡喃半乳糖苷(IPTG)誘導(dǎo)表達(dá)、分離純化發(fā)現(xiàn)該蛋白的分子量約為43kD,等電聚焦電泳結(jié)果表明,該蛋白的等電點(diǎn)為6.1,HPLC檢測(cè)說(shuō)明合成方向的酶活性為每毫克蛋白每分鐘4.5μmol,DNTB與同型半胱氨酸顏色反應(yīng)測(cè)定水解方向的酶活性為每毫克蛋白每分鐘0.7μmol,根據(jù)上述特性可以確定草菇冷誘導(dǎo)基因Cor3所編碼的蛋白是S-腺苷同型半胱氨酸水解酶(SAHH)。
【Abstract】 The full-length cDNA sequence of Volvariella volvacea Cor3 gene was digested with EcoRI 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 chromatogr...
【關(guān)鍵詞】 S-腺苷同型半胱氨酸水解酶基因； 草菇； 冷誘導(dǎo)基因；
【Key words】 S-adenosyl-L-homocysteine hydrolase gene； Volvariella volvacea； cold induced gene；
【基金】 草菇基因組和功能基因組數(shù)據(jù)庫(kù)共享平臺(tái)建設(shè)(編號(hào):10dz2212400);上海市科學(xué)技術(shù)委員會(huì)重點(diǎn)項(xiàng)目的部分研究?jī)?nèi)容
【分類號(hào)】S646.13