大白菜cDNA-AFLP分析体系的优化  

Optimization of cDNA-AFLP Analysis System for Chinese Cabbage

作　　者：郭莹[1] 杨晓云[2] 张清霞[2] 司朝光[2] 张淑霞[2] 王媛[2] 

机构地区：[1]青岛农业大学园林园艺学院,青岛266109 [2]青岛市农业科学研究院,青岛266100

出　　处：《分子植物育种》2010年第5期1003-1007,共5页Molecular Plant Breeding

基　　金：国家十一五科技支撑计划项目(2008BADB1B01-3)资助

摘　　要：本研究以高感、高抗大白菜小黑点病的大白菜叶柄为试材,对影响cDNA-AFLP分析体系的几个关键因素进行分析,建立了适宜大白菜的cDNA-AFLP分析体系,并得到了较为清晰可辨的cDNA-AFLP图谱。结果表明:试剂盒法提取的NA较完整,纯度较高;400ng的cDNA利用FastDigest EcoRⅠ和Fast-Digest MseⅠ进行快速双酶切50min,酶切充分;在20μL的体系中,连接产物取原液作为预扩增反应的模板,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,扩增结果最佳;MBI2×PC Master Mixture反应体系满足扩增反应的需要,不需要另外摸索反应条件,节省了试验成本;选用64对AFLP引物进行扩增,筛选出具有多态性条带丰富的AFLP引物45对。本研究为利用cDNA-AFLP分析大白菜小黑点病的的发病机理奠定了基础。In this paper,we analyzed the key facters affecting cDNA-AFLP,established cDNA-AFLP analysis system for Chinese cabbage by employing petioles with high resistance and high susceptibility to petiole spot in Chinese cabbage,and obtained a distinct electrophoretogram of cDNA-AFLP.The results indicated that RNA extracted by kit had high purity and integrity;400 ng cDNA was double digested completely by FastDigest  EcoRⅠ and FastDigest MseⅠfor50 min;Optimal results was obtained in following ways：an initial 35-cycle preamplification PCR by using original ligation products as template and the preamplification products diluted to 30 times for selective amplification in 20 μL reaction volume.MBI＇s 2×PCR Master Mixture reaction system meet the needs for amplification.It is cost saving without having to explore other reaction conditions.45 pairs of AFLP primerwith high polymorphic bands were screened from 64 pairs of AFLP primer.This research provided insights into pathogenic mechanism of petiole spot in Chinese cabbage.

关 键 词：大白菜 CDNA-AFLP 分析体系 小黑点病 

分 类 号：S634.1[农业科学—蔬菜学]