苎麻茎皮cDNA文库的构建

Construction of bark cDNA library of Ramie(Boehmeria nivea(L.)Gaud)

以生长中期的苎麻茎皮为材料,提取总RNA,经纯化mRNA后,合成双链cDNA。双链cDNA加上EcoRI-SmaI接头后,用限制酶NotI酶切并除去小于400bp的短链cDNA,长的cDNA片段连接到EcoRI-NotI酶切载体pAP3neo上,获得滴度为2.6×104的苎麻cDNA文库,插入片段大部分分布在500bp～1500bp之间。该文库的建立为苎麻表皮功能性新基因的发现奠定了基础。

Abstract： Total RNA was extracted from the ramie（ Boehmeria nivea（L. ） Gaud） barks harvested at the mid - growing stage, and mRNA was purified. After double - stranded cDNAs synthesis, EcoRI - Sma I adaptor were added to blunted cDNA and digested with Not I, and fractionated by spin column. The fragments longer than 400bp were ligated into the pAP3neo Predigested Vector. The cDNA library consisted of 2. 6 × 10^4pfu/ml, a majority of inserts size ranged between 500bp and 1500bp. This constructed cDNA library provided an essential resource for future functional genomic analysis of ramie（ Boehmeria nivea （L.）Gaud）.