摘要
为提高缩叶肉质盐生植物海蓬子总RNA和mRNA的质量，将改良CTAB法与Tripure试剂盒方法相结合，从300mg海蓬子发芽后20d的幼苗中成功分离和纯化了高纯度的总RNA和mRNA。并以无NaCl处理为Driver，以200mmol／LNaCl处理24h、48h、72h的幼苗为Tester，通过cDNA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200～1300bp的cDNA序列，pGEM-T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2，6×10^7和96％，共收集阳性克隆12000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。两种RNA提取方法的结合有效提高了多汁多色素海蓬子RNA的质量，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抗盐基因奠定了基础。
Pure total RNA and mRNA were successfully isolated from succulent halophyte, Salicornia bigelovii Tort. , using a combining method of modified CTAB and commercial kit （Roche）. Following the synthesis of double cDNA, suppression subtractive hybridization （SSH） was carried out with the mixed cDNA from 20-day-old seedlings treated with 200 mmol/L NaC1 for 24 h,48 h, and 72 h as ‘ Tester＇ and those treated without NaC1 as ‘ Driver＇. Then a series of differential cDNAs sized from 200 to 1 300 bp were powerfully enriched. The linkage of cDNA with pGEM-T vector and white-blue screening indicated that the content of library clones and recombinant rate reached 2. 6 × 107 and 96% respectively. The EcoR Ⅰ 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 salt tolerance.
出处
《江苏农业学报》
CSCD
北大核心
2006年第2期113-116,共4页
Jiangsu Journal of Agricultural Sciences
基金
江苏省自然科学基金(BK2003201)
江苏省农业科学院基金(6110530)
