缺刻缘绿藻cDNA文库的构建

Construction of cDNA library of a green microalga,Myrmecia incisa

缺刻缘绿藻（Myrmecia incise）富含花生四烯酸（arachidonic acid，AA），是迄今为止所知道的花生四烯酸含量最高的藻类。采用Trizol法提取总RNA，然后按照Clontech公司的SMART cDNA文库构建的方法，构建了缺刻缘绿藻的cDNA文库，测得的原始文库滴度为6×10^6pfu／mL，随机挑选文库的阳性单克隆进行PCR鉴定，扩增出的片段主要集中在0．5-1．5kb，平均插入片段长度约为1kb，文库的重组率达95．83％，说明本实验所构建的cDNA文库质量合格。

The freshwater green microalga Myrmecia incisa was isolated from Mt. Tateyama, Japan. It is well-known by far that this microalga is the richest source of one polyunsaturated fatty acid （ PUFA）, arachidonic acid （20： 4ω6, AA）. Total RNA was isolated from this alga by Trizol method, and the cDNA library of M. incisa was constructed using SMART cDNA Library Construction Kit, Clontech, Inc. The titer of unampified library reached 6 ×10^6 pfu/mL, and the recombination rate was about 95. 83%. PCR amplification of randomly selected positive clones illustrated the inserted cDNA fragments ranged from 0.5 kb to 1.5 kb, and the average length was about 1 kb. All these results suggested that the cDNA library was successfully constructed.