水稻叶片cDNA-AFLP技术体系的建立和优化

Establishment and Optimization of cDNA-AFLP Reaction System for Rice Leaves

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。

In this paper, we analyzed the key factors affecting cDNA-AFLP, established cDNA-AFLP analysis system for rice by employing petioles with high resistance and high susceptibility to petiole spot in rice, and obtained a distinct electrophoretogram of cDNA-AFLP. The results indicated that RNA extracted by centrifugal columnar （RNeasy Plant Mini Kit, Qiagen） method had high purity and integrity;eDNA reversed by M-MLV RTase cDNA Synthesis kit （TAKARA）was digested with EcoR I enzyme under 37 ~C for 2 h and Mse I enzyme under 65 ~C for 2 h and linked under 4 ~C for 13 h. Optimal results was obtained in following ways：an initial 35-cycle preamplification PCR by using 10-fold dilution of ligation products as template and the preampli- fication products diluted to 30 times for selective amplification in 20 p^L reaction volume. By the separation of 6% PAGE gel and rapid silver staining,50 pairs of AFLP primer with high polymorphic bands were screened from 64 pairs of AFLP primer. This research provided insights into further study of heat-tolerant gene in flee.