扁蓿豆冷诱导基因转录差异cDNA-AFLP反应体系的构建  

Establishment of cDNA-AFLP Reaction System of Cold-induced Genes Transcript Difference for Medicago ruthenica

作　　者：张宇[1] 石凤翎[1] 高霞[1] 蔡丽艳[2] 

机构地区：[1]内蒙古农业大学生态环境学院,内蒙古呼和浩特010019 [2]中国农业科学院草原研究所,内蒙古呼和浩特010010

出　　处：《中国草地学报》2013年第2期13-18,共6页Chinese Journal of Grassland

基　　金：内蒙古应用技术研究与开发资金项目--退化及受损生态系统治理恢复与资源综合利用技术研究"抗逆优良乡土草种的选育研究"课题;内蒙古农村领域科技计划项目(20091401)

摘　　要：以0℃低温处理3～5叶期扁蓿豆的幼苗嫩叶为试验材料,对影响其cDNA-AFLP反应体系的各个关键因子进行探讨和优化,建立适宜的扁蓿豆cDNA-AFLP扩增体系,并对扁蓿豆冷诱导的差异表达进行检测。结果表明,利用MseⅠ/EcoRⅠ分步酶切连接,以4μl稀释20倍的预扩产物作为模板、1.6μl dNTP、0.4μlTaq DNA酶、2μl10×PCR Buffer获得的选择性扩增效果较好,条带清晰稳定。应用该反应体系扩增出的扁蓿豆冷诱导差异片段有五种表达类型,为扁蓿豆抗寒基因转录水平差异表达的研究奠定了基础。The cDNA amplified fragment length polymorphism （cDNA-AFLP） reaction system of Medicago ruthenica was established and the genes expression related with cold tolerance were tested after optimizing several key factors affecting cDNA-AFLP analysis using the leaves at 3 -5 leaf stage treated with 0℃. The results showed that the amplified products resulted from this reaction system was better, cDNA was digested by Mse I and EcoR I step by step, 20 times-diluted pre-amplification 4μl, dNTP 1.6μl, Taq enzyme 0.4μl, 10 X PCR Buffer 2μl, and the fragments were clear and stable. The amplified cold induced differential fragments of Medicago ruthenica can be divided into five types, this experiment laid a base for studies on differential expression of gene relate to cold tolerance in transcript level.

关 键 词：扁蓿豆 冷诱导 CDNA-AFLP 差异表达 

分 类 号：S541[农业科学—作物学]