目的：为构建红花的遗传连锁图谱和功能基因的研究, 探讨影响红花cDNA扩增片段长度多态性(cDNA amplified fragment length polymorphism, cDNA-AFLP)的各种因素, 建立并优化红花cDNA-AFLP反应体系。方法：以红花新鲜花瓣为材料, 针对其内含物特殊性对Trizol法加以改进提取RNA, 采用无RNase H活性的鼠源反转录酶(M-MLV RTase)结合置换合成法反转录双链cDNA;两步法酶切与连接, 优化酶切时间;连接产物和预扩产物分别设置不同稀释倍数扩增并对选扩反应体系略微调整后, 用6%的聚丙烯酰胺凝胶(PAGE)电泳分离, 银染检测。结果：改进Trizol法得到的红花总RNA样品较为完整, 纯度较高, 可直接用于双链的合成;经无RNase H活性反转录酶结合置换合成法得到高质量的cDNA模板。建立优化后的红花cDNA-AFLP体系为:250 ng cDNA 37 ℃6 h经限制性内切酶MseI和EcoRI完全酶切, 16 ℃连接12 h;连接产物最佳稀释倍数为10倍;预扩产物稀释为150倍;PAGE电泳得到清晰、稳定、分辨率较高的多态性条带。结论：本研究建立的反应体系适用于红花功能基因的cDNA-AFLP研究。

Objective: To construct for the heredity linkage map and to study the functional gene research of Carthamus tinctorius, the factors affecting cDNA amplified fragment length polymorphism (cDNA-AFLP) of C. tinctorius were investigated with developing and optimizing the eDNA-AFLP reaction system. Methods: Improved Trizol method was used to extract RNA from compounds in new petals specific to safflower. With thehelp of M-MLV RTase without RNase activity combined with replacement synthesis method, double-stranded eDNA was synthesized from total RNA；eDNA was digested by restriction enzyme MseI/EcoRI and ligated bytwosteps. Then the productswere provided for preamplification and selected amplification of different concentration gradients. After tiny modifications of system concentration, finally PAGE electrophoresis and silver-staining were performed. Results: High purity and integrated total RNA for later eDNA synthesis were obtained and high quality cDNA was synthesized with the helpof M-MLV RTase without RNase activity combined with replacement synthesis method. The cDNA-AFLP reaction system in C. tincto Hus was as follows：250ng integrity cDNA was digested thoroughly at 37 ℃ for 6 h, and ligated 12 h at 16 ℃. Furthermore, the sample dilution multiplication was 10 fold for pre-amplification and 150 fold for selected amplification under the proper system concentration. According to the above reaction system, the polymorphous strips with high resolution powerin PAGE electrophoresis were clear and stable. Conclusion: The cDNA-AFLP reaction system established and optimized in this experiment is suitable for the functional gene analysis of C. tinctorius.

国家自然科学基金资助项目(30772734);上海市基础研究重点项目(043919313)