利用酿酒酵母表达氯化钠胁迫下旱柳全长cDNA文库  

Expressed salt stress induced full length-cDNA library of Salix matsudana using yeast Invsc1

作　　者：杨晔[1,2] 蒋晶[2] 乔桂荣[2] 周婧[2] 陈银[2] 何正权[1] 李海营[2] 卓仁英[2] 

机构地区：[1]三峡大学生物技术研究中心/天然产物研究与利用湖北省重点实验室,湖北宜昌443002 [2]中国林业科学研究院亚热带林业研究所,浙江富阳311400

出　　处：《浙江林学院学报》2009年第4期473-478,共6页Journal of Zhejiang Forestry College

基　　金：非盈利性科研院所基金资助项目(RISF060702)

摘　　要：为了研究旱柳Salix matsudana高耐盐无性系I-32的耐盐机制,分离其耐盐相关基因,实验以筛选出的高耐盐旱柳无性系I-32为材料,盐胁迫后提取总核糖核酸(RNA),纯化mRNA,利用改良SMART(switching mechanism at 5’end of RNA transcript)技术合成了全长cDNA,回收500bp以上cDNA大片段克隆到大肠杆菌Escherichia coli和酵母穿梭载体pYES2.1中,建成旱柳全长cDNA文库,对随机挑取的阳性克隆进行聚合酶链式反应(PCR)鉴定,插入片断平均值为1000bp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。通过尿嘧啶缺陷型培养基添加80～130g·L-1氯化钠筛选文库,与野生型酵母菌株相比,转化子的耐盐能力提高。研究结果为克隆与旱柳抗旱相关基因奠定了基础。The studies of Salix matsudana on salt-resistance molecular mechanism and searching new salt- tolerant relation genes are limited at present. It is necessary to develop drought-tolerant gene using this valuable tree resource, so a salt stress induced full-length cDNA Library of Salix matsudana was constructed using Clontech Laboratories, Inc. SMART cDNA Library Construction Protocol. Total RNA was extracted from S. matsudezm under 100 mmol.L^-1 NaC1 stress for 24 h. After synthesizing the first-strand cDNA, the double-strand cDNA was amplified with the long-distance Polymerase Chain Reaction （LD-PCR）. The result showed that insertion fragment was about 1 000 bp length in the library by PCR. The full-length cDNA frag- ment was linked to yeast vector p YES2.1 by p YES2.1 TOPO TA Expression Kit; then the linked product was transferred into yeast Invscl. Finally, clones were selected by SC-U medium containing 80 - 130 g.L＆-1 NaCl. The experiment shows that the salt-tolerance of transformants yeast improved greatly compared with the wild-type yeast. The results provide an important tool for the study of cloning new salt-tolerance relation genes and the salt tolerance mechanisms of the S. mats udana. [Ch, 6 fig. 17 ref. ]

关 键 词：植物学 旱柳 CDNA文库 耐盐性 酵母 抗旱相关基因 

分 类 号：S718.43[农业科学—林学] Q789[生物学—分子生物学]