萘胁迫下水稻幼苗抑制差减杂交cDNA文库构建

Construction of cDNA library of rice seedlings roots under nap stress based on suppression subtractive hybridization

从200mg／№萘胁迫水稻幼苗根系中成功分离和纯化了高纯度的总RNA和mRNA，并以无萘处理为Driver，以200mg／Kg的幼苗为Tester，通过eDNA双链的合成及两次PCR扩增，富集到两组处理中差异表达的大小在200～800bp的eDNA序列，pGEMT的连接和蓝白斑筛选表明杂交文库滴度和重组率分别达到1．5×10^6pfu／mL和96％，共收集阳性克隆8000个，形成了抑制消减杂交的质粒cDNA文库。随机挑取酶切质粒的电泳分析表明，载体中均有插入片段。高效抑制消减杂交文库的建立为进一步分析基因的差异表达及分离萘胁迫下抗性基因奠定了基础。

Pure total RNA and mRNA were successfully isolated from rice seedlings roots Following the synthesis of double cDNA, suppression subtractive hybridization （SSH） was carried out with the mixed cDNA from seedlings treated with 200 mg/Kg Nap as＇ Tester＇ and those treated without Nap as＇ Driver＇. Then a series of differential cDNAs sized from 200 to 800 bp were powerfully enriched. The linkage of cDNA with pGEMT vector and white blue screening indicated that the content of library clones and recombinant rate reached 1.5 × 10^6 and 96% respectively. The EcoR digested of random recombinant plastids showed that each had been inserted by cDNA sequences. High efficient construction of SSH cDNA library paved the way for gene expression analysis and discovery of new genes contributing to Nap tolerance.