青枯菌诱导的花生根全长cDNA文库的构建  

Construction of Peanut Root Full-length cDNA Library Induced by Ralstonia solanacearum

作　　者：张冲[1,2] 曾建斌[1] 陈华[1] 姜宝杰[1] 贺小彦[1] 庄伟建[1] 

机构地区：[1]福建农林大学福建省作物分子与细胞生物学重点实验室,福建福州350002 [2]福建农林大学生命科学学院,福建福州350002

出　　处：《热带作物学报》2013年第10期1941-1946,共6页Chinese Journal of Tropical Crops

基　　金：花生抗黄曲霉基因工程及育种研究(No.2008DFA31450);特色植物功能基因组学研究与应用(No.2013AA102600)

摘　　要：以优质抗青枯病花生品种闽花8号为材料,在苗期利用灌根法接种花生青枯菌,分不同时期取根,利用改良的CTAB-LiCl法提取接种和非接种的混合RNA,采用SMART（Switching Mechanism At 5＇end of RNATranscript）技术合成双链cDNA,经SfiⅠ酶切后胶回收纯化双链cDNA,连接到质粒载体pDNR-LIB上,电击转化大肠杆菌DH5α感受态细胞,成功构建了青枯菌诱导的处理和对照混合全长cDNA文库.经鉴定,初级文库库容为1.78×10 cfu/mL,重组率达到99％以上,插入片段集中在750～2 500 bp之间,平均长度约为1 300 bp,经扩增后的文库滴度为2.97×109 cfu/mL.随机挑取部分克隆测序,利用生物信息学软件进行初步分析获得一些EST数据.说明得到了高质量的全长cDNA文库,这为筛选和克隆青枯菌诱导的胁迫相关基因提供了资源基础.The roots of high resistant peanut variety Minhua 8 were inoculated by Ralstonia solanacearum and harvested at different time points.Total RNA was extracted by improved CTAB-LiC1 method.dscDNA was synthesized based on SMART system.The purified double strand cDNA was ligated to Sfi Ⅰ digested vector pDNR-LIB,and transformated into DH5α by electroporation.A successful full-length cDNA library was constructed,which had a primary library titer of 1.78×106 cfu/mL.Colony PCR results showed that the inserts varied from 0.75 to 2.50 kb with average size larger than 1 000 bp and 99％ of recombinant percentage,and the amplyfied titer was 2.97×109 cfu/mL.Part of the clones were randomly selected and sequenced.Some EST data were got after preliminarily bioinformatic analysis.The results indicated that the library was of high quality,which provided specific resource base for further cloning and screening related stressed genes induced by R.solanacearum.

关 键 词：花生 青枯病 SMART技术 CDNA文库 

分 类 号：S565.2[农业科学—作物学] Q943.2[生物学—植物学]