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以高温和常温处理的水稻幼苗叶片为材料,对影响cDNA-AFLP分析体系的关键因素进行了分析,建立了适宜水稻的cDNA-AFLP分析体系,并得到了清晰可辨的cDNA-AFLP指纹图谱。结果表明:离心柱式试剂盒提取的RNA较完整,纯度较高;双链逆转录试剂盒形成的cDNA经EcoRⅠ(37℃,2 h)和MseⅠ(65℃,2 h)完全酶切后,4℃连接过夜;20μL的体系中,连接产物稀释10倍液5.0μL作为预扩增的模板,并且预扩增反应的循环数为35,预扩增产物稀释20倍作为选择性扩增的模板,扩增结果较佳;6%PAGE分离快速银染法显示,64对选择性扩增引物中筛选出多态生条带丰富的AFLP引物50对。本研究为利用cDNA-AFLP分析水稻抗高温基因进行深入的研究奠定了基础。
The key factors influencing the cDNA-AFLP analysis system were analyzed using rice seedling leaves treated at high temperature and room temperature. The cDNA-AFLP analysis system suitable for rice was established and the fingerprint of cDNA-AFLP was obtained clearly. The results showed that the RNA extracted from the spin column kit was more complete and of higher purity. After the cDNAs were double digested with EcoRⅠ (37 ℃, 2 h) and MseⅠ (65 ℃, 2 h) 4 ° C overnight; in a 20 μL system, 5.0 μL of the 10 μL dilution of the ligation product was ligated as a template for pre-amplification and the number of cycles of the pre-amplification reaction was 35 and the pre-amplification product was diluted 20-fold as a template for selective amplification , And the amplification result was better. The rapid silver staining with 6% PAGE showed that there were 50 pairs of polymorphic AFLP primer pairs amplified by 64 pairs of selective amplification primers. This study lays the foundation for the further study of using cDNA-AFLP to analyze the high temperature resistance genes in rice.