甘草根cDNA文库构建与分析

目的：构建甘草cDNA文库，为甘草功能基因的研究以及筛选、克隆与甘草次生代谢产物合成途径相关基因奠定基础。方法：以LiCl沉淀法提取根组织总RNA，置换合成法合成双链cDNA，末端补平，连接EcoR I并磷酸化，以pBlueScriptⅡ为载体并电转化感受态细胞E.coli DH10 β，测定文库的克隆数、重组率，PCR 测定插入片断大小。同时，对文库进行随机测序并在GenBank中进行同源性检索和功能预测。结果：经鉴定文库的库容量为1.15×10⁷，重组率为98.2％，插入片段在0.5～4.8 kb，平均片段长度大于1.0 kb；经随机测序，获得126条EST，BLAST分析表明，大多数EST与豆科植物以及拟南芥、水稻等植物基因序列具有较高的同源性，在86条已知功能基因中，主要为细胞代谢、抗逆性、生长抑制及休眠相关的基因，其余40条EST为未知功能基因。结论：构建的甘草根cDNA文库库容量大、重组率高且插入的cDNA片段较长，能够满足甘草功能基因的研究。

Construction and analysis of root cDNA library in Glycyrrhiza uralensis

OBJECTIVE: To screen and isolate secondary metabolite biosynthesis-related gene for establishing the foundation of functional gene research, we construct a cDNA library of Glycyrrhiza uralensis. METHOD: Total RNA was isolated from G. uralensis using the method of lithium chloride sedimentation. Double strand cDNA was joined into pBlueScript II vector. The number of clones, recombinant rate and length of insert fragments were determined. RESULT: The capacity of the original library was 1.15 x 10(7) with a recombinant rate of 98.2% and the inserted cDNA fragments ranged from 0.5 to 4.8 kb. 126 ESTs through random sequencing were obtained. The most homological proteins came from leguminous plants, including Arabidopsis thaliana, Oryza sativa, and so on. Most of the proteins were related to genes linking cell matabolism, resistance, growth retardation and dormancy. CONCLUTION: The library has enough capacity, high recombinant rate and long insert fragment for the further study.