低温诱导东乡野生稻幼苗期叶片cDNA文库的构建和鉴定

Construction and Identification of a cDNA Library from Dongxiang Wild Rice Induced by Low Temperature

从8℃低温诱导的东乡野生稻幼叶中提取总RNA,以总RNA为模板,利用SMART技术合成第1链,再经LD-PCR扩增合成第2链后,将获得的双链cDNA与双元表达载体YPL3连接,重组质粒电转入大肠杆菌,成功构建了东乡野生稻cDNA文库。经测定,原始文库滴度平均为4.4×107cfu/mL,平均插入片段约1kb,重组率为100%,符合cDNA文库构建要求,为后续利用酵母功能互补技术筛选东乡野生稻有利基因奠定了基础。

Total RNA was exacted from the seedling leaves of Dongxiang wild rice after treatment of low temperature at 8℃. The first and second cDNA strands were synthesized by SMART method and LD-PCR amplification, and the double strand cDNA（ ds cDNA） fragments were ligated to the binary expression vector YPL3. The recombinant plasmids were transformed into the E. coli, and the cDNA library of Dongxiang wild rice was successfully constructed. The results showed that the titer of the unamplified cDNA library was 4.4×10^7cfu/mL, the average length of inserts was about 1 kb and the recombination rate was 100% , which indicates that this library could be used for yeast functional complementation screening and cloning of favorable genes in Dongxiang wild rice.