矿山型东南景天cDNA表达文库构建与耐镉基因筛选

以筛选出的镉超积累矿山型东南景天为材料,经镉胁迫后提取总RNA,纯化mRNA,利用改良SMART技术合成了全长cDNA,回收500 bp以上cDNA大片段克隆到改造过的大肠杆菌/酵母穿梭载体pYES2.0G中,建成东南景天镉全长cDNA文库,对随机挑取的阳性克隆进行PCR鉴定,插入片断大小在1 000 bp左右,说明所构建的文库达到了用于目的基因分离筛选和表达的建库要求,为将来筛选克隆与东南景天抗重金属相关基因奠定了基础。...

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Bibliographic Details
Published in林业科学研究 Vol. 25; no. 3; pp. 332 - 338
Main Author 刘明英 乔桂荣 蒋晶 邱文敏 卓仁英
Format Journal Article
LanguageChinese
Published 中国林业科学研究院亚热带林业研究所,浙江富阳311400 2012
林木遗传育种国家重点实验室,北京100091
Subjects
cDNA文库
东南景天
耐镉
酿酒酵母
耐镉
东南景天
酿酒酵母
cDNA文库
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ISSN1001-1498
DOI10.3969/j.issn.1001-1498.2012.03.010

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Summary:以筛选出的镉超积累矿山型东南景天为材料,经镉胁迫后提取总RNA,纯化mRNA,利用改良SMART技术合成了全长cDNA,回收500 bp以上cDNA大片段克隆到改造过的大肠杆菌/酵母穿梭载体pYES2.0G中,建成东南景天镉全长cDNA文库,对随机挑取的阳性克隆进行PCR鉴定,插入片断大小在1 000 bp左右,说明所构建的文库达到了用于目的基因分离筛选和表达的建库要求,为将来筛选克隆与东南景天抗重金属相关基因奠定了基础。
Bibliography:A cadmium stress induced full-length cDNA Library from the Sedum alfredii was constructed by using improved SMART cDNA library construction protocol.Total RNA were extracted from S.alfredii under cadmium stress.After synthesizing the first-strand cDNA using limited amount of total RNA,the double-strand cDNA was amplified with long-distance PCR method.The full-length cDNA fragment was linked to versatile vector pYES2.0G,then the linked product was transferred into yeast INVSc1.The clones were randomly selected to sequence and bioinformatics analysis was carried out.The inserted fragments in the library are about 1000 bp length.This full-length cDNA Library provides a important tool for the study of the salt tolerance mechanisms of S.alfredii.
11-1221/S
Sedum alfredii; cDNA library; Cd tolerance; yeast INVSc1
LIU Ming-ying,QIAO Gui-rong,JIANG Jing,QIU Wen-min,ZHUO Ren-ying,（1.Research Institute of Subtropical Forestry,Chinese Academy of Forestry,Fuyang 311400,Zhejiang,China; 2.State Key Laboratory of Tree Genetics
ISSN:1001-1498
DOI:10.3969/j.issn.1001-1498.2012.03.010