利用改进的Oligo-Capping法构建甘蔗茎全长cDNA文库

Construction of Full-length cDNA Library for Sugarcane Stem by Optimized Oligo-capping

Oligo-Capping法是目前全长cDNA文库构建的重要方法之一。笔者在引进、消化和吸收的基础上,通过对特异引物和接头序列的设计,及cDNA扩增反应条件的优化,对该方法进行了一些切实可行的改进,形成了一套简便易行的技术体系。利用本研究中改进的Oligo-Capping法,成功地构建了甘蔗茎全长cDNA文库。综合评价结果表明,本研究中所构建的甘蔗茎全长cDNA文库的库容大于2.5×106cfu,重组率为91%,全长率高达88.9%。本文库的构建为进一步甘蔗茎中全长基因的克隆和功能基因的表达分析奠定了基础。

Oligo-capping is one of the most important methods in the construction of full-length eDNA library. In order to discover new genes in a throughput manner, the Oligo-capping method published previously was modified to raise the efficiency in the construction of full-length eDNA library, and a quick and simple technical system for constructing a full-length eDNA library was then successfully established. Using the optimized protocol, we successfully constructed a full-length eDNA library for sugarcane stem, which contained 2.5×10^6 cfu with 91% recombinant phages. The result of sequence analysis indicated that the full-length cDNA proportion amounted to as high as 88.9%, while that of cDNA library constructed with the traditional oligo-capping method only ranged from 60% to 70%. The library constructed had laid a foundation for the full-length gene cloning and expression analysis of functional genes in sugarcane stem