两步扩增法在玉米抗大斑病相关基因片段分离中的应用  

The application of two-step amplification method in separation of expressed gene fragments in corn resistant to Setosphaeria turcica

作　　者：谷守芹[1] 王海祥[2] 范永山[1] 韩建民[1] 董金皋[1] 

机构地区：[1]河北农业大学真菌毒素实验室,河北保定071001 [2]保定师范专科学校,河北保定071000

出　　处：《河北农业大学学报》2005年第1期53-55,68,共4页Journal of Hebei Agricultural University

基　　金：河北省自然基金项目(302333)

摘　　要：对一步扩增法和两步扩增法在DDRT-PCR中的扩增效率进行了比较,结果发现,两种方法得到的片段大小都在150～1500bp之间,但条带数量和清晰度不同.一步扩增法得到扩增条带为30～45条,条带较模糊,两步扩增法得到的扩增条带为40～60条,条带清晰.一步扩增法获得10条玉米抗大斑病差异表达条带,只有55条带中的cDNA能够成功回收并产生有效扩增,差异条带回收率53.9%,而两步扩增法获得了148条差异条带,其中138条带都能得到有效回收和扩增,回收率达93.2%.因此,步扩增法更有利于差异条带的回收和二次扩增.The amplification efficiency of one-step amplification method and the two-step amplification method in differential display reverse PCR (DDRT-PCR) were compared. It was found that the length of the amplified fragments by the two methods was all about 150～1000 bp,but the definition and the number of amplification band was different.30～45 bands were obtained by one-step amplification method, but the bands obtained by two-step were about 40～60.The bands obtained by two-step amplification method were much more legible than those obtained by one-step amplification method.102 differentially expressed bands related to the corn resistance to Setosphaeria turcica were acquired by one-step amplification method, in which 55 cDNA bands were successfully recovered, and the recovery percentage of the bands was about 53.9% while by two-step amplification method 148 amplification bands were acquired, in which 138 cDNA bands were successfully recovered, and the recovery percentage was 93.2%.In conclusion, the two-step amplification method was much more suitable for the mRNA differential display.

关 键 词：玉米 两步扩增法 玉米大斑病 抗病相关基因 

分 类 号：S435.13[农业科学—农业昆虫与害虫防治]