芍药种子cDNA-AFLP体系的建立

Establishment of c DNA-AFLP System of Paeonia lactiflora Seed

以芍药品种‘粉玉奴’与‘粉中楼’的杂交种子为试材,通过对影响cDNA-AFLP反应的重要因素进行优化,建立了适合于芍药种子的cDNA-AFLP分析体系,并对打破上胚轴与下胚轴休眠前后的种子基因表达差异进行了初步分析。结果表明:使用RNAprep Pure植物总RNA提取试剂盒提取的RNA较完整;cDNA利用EcoRⅠ和MseⅠ进行双酶切37℃4 h,65℃20 min,酶切充分;连接产物稀释10倍用于预扩增;预扩增产物稀释10倍,取5μL作为选择性扩增的模板,得到了较为清晰可辨的cDNA-AFLP谱带;打破下胚轴休眠前后约有3700条差异条带,打破上胚轴休眠前后约有1600条差异条带。

The research established a cDNA-AFLP analysis system which suited Paeonia lactiflora seed by using hybrid seeds of P.‘Fenyunu’ and P.‘Fenzhonglou’ as materials and optimizing the important factors which affect the reaction of cDNA-AFLP. In addition,we made a preliminary analysis on the expression difference of gene under the conditions of before and after breaking epicotyl and hypocotyl dormancy. The results indicated that total RNA was isolated from Paeonia lactiflora seeds using the RNAprep Pure Plant Kit according to the manufacturer's instruction. Doubled-stranded c DNA was digested completely with Eco RⅠ and MseⅠat 37 ℃ for 4 h and at 65 ℃ for 20 min. The ligated products should be diluted 10 times with sterilized distilled water for the pre-amplification. Then relatively clear and differentiable bands were obtained after the pre-amplified products were diluted 10 times,and 5μL were taken as a template for selective amplification. About 3700 differential bands were visualized between before and after breaking hypocotyl dormancy and about 1600 differential bands were visualized between before and after breaking epicotyl dormancy.