增强UV-B辐射对小麦幼苗基因组DNA的影响及RAPD分析体系的建立  

Effect of Enhanced UV-B Radiation on Genomic DNA in Wheat Seedling and Establishment of RAPD Analysis System

作　　者：李苗[1] 高丽美[1] 李永锋[1] 韩榕[1] 

机构地区：[1]山西师范大学生命科学学院,临汾041004

出　　处：《生物技术通报》2010年第5期87-92,共6页Biotechnology Bulletin

基　　金：国家自然科学基金(30671061);山西省自然科学基金(2008011059-1;20041101)

摘　　要：采用改进的CTAB法提取小麦总基因组DNA,并以之为模板对RAPD反应体系中的一些重要参数进行梯度试验,建立了一套适合本研究的最佳反应体系。即25μL反应体系:模板DNA 20 ng、引物浓度0.5μmol/L、dNTPs浓度200μmol/L、Taq酶0.750 U。反应程序:94℃预变性2 min,94℃变性30 s,38℃退火30 s,72℃延伸30 s,30个循环,72℃延伸10min,4℃保存。此外,利用扩增结果稳定的引物对正常光照组及增强UV-B辐射处理组的小麦DNA进行RAPD分析。结果表明,增强UV-B辐射处理组的DNA,经引物S22、S40扩增后分别出现了分子量为2 144 bp和2 082 bp的差异条带,这能否从一定程度上揭示UV-B对植物造成损伤的分子生物学机制,还有待进一步的研究。The genomic DNA extracted by advanced CTAB method from the fresh leaves of wheat seedlings,which could be used for RAPD analysis as template. During the research,the main parameters in polymerase chain reaction were studied by setting gradient in order to obtain an optimal system for RAPD analysis. The proposal improved was as follows,25 μL reaction system containing 20 ng template,0.5 μmol/L primers,200 μmol/L dNTPs,0.75 U Taq polymerase. The optimal amplification program was as follows ,2 min at 94℃ ,followed by 30 cycles of 30 s at 94℃ ,30 s at 38℃ ,30 s at 72℃ ,and then a final extension at 72℃ for 10 min. For further study, the PCR products can be stored in 4℃ for a long time. Besides, the damaged wheat seedlings induced by enhanced ultraviolet-B（ 10.08 kJ · m ^-2 . d ^-1 ） radiation and the control ones were made for RAPD analysis using the screened random primers which can obtain reproducible results. The result shows that two specific bands of 2,144bp and 2,082bp separatedly occurred in the UV-B treatments amplified with S22 and S40. The problem about the molecular mechanism of the damage induced by UV-B still needs further study.

关 键 词：小麦 UV—B辐射 CTAB法 RAPD分析 差异条带 

分 类 号：S512.1[农业科学—作物学]