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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 extracted from young leaves of Dongxiang wild rice induced by low temperature of 8 ℃. The first strand was synthesized by SMART technique using total RNA as a template. After the second strand was synthesized by LD-PCR amplification, the double-stranded cDNA The binary vector YPL3 was ligated and the recombinant plasmid was transformed into Escherichia coli. The Dongxiang wild rice cDNA library was successfully constructed. The results showed that the titer of the original library was 4.4 × 107 cfu / mL on average, about 1 kb in average insert and 100% in recombination rate, which conformed to the requirement of cDNA library construction. This laid the foundation for further screening of Dongxiang Oryza sativa by yeast functional complementation.