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园艺学报 ›› 2015, Vol. 42 ›› Issue (3): 576-584.doi: 10.16420/j.issn.0513-353x.2014-0699

• 研究报告 • 上一篇    下一篇

芍药种子cDNA-AFLP体系的建立

孙晓梅,杨盼盼,陆秀君,周文强,王 丹,杨宏光   

  1. 1沈阳农业大学林学院,沈阳 110866;2沈阳市植物园,沈阳 110163
  • 出版日期:2015-03-25 发布日期:2015-03-25
  • 基金资助:

    国家自然科学基金项目(31240028,31470696)

Establishment of cDNA-AFLP System of Paeonia lactiflora Seed

SUN Xiao-mei1,*,YANG Pan-pan1,LU Xiu-jun1,ZHOU Wen-qiang2,WANG Dan2,and YANG Hong-guang1   

  1. 1College of Forestry,Shenyang Agricultural University,Shenyang 110866,China;2Shenyang Botanical Garden,Shenyang 110163,China
  • Online:2015-03-25 Published:2015-03-25

摘要: 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5 μL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3 700条差异条带,打破上胚轴休眠前后约有1 600条差异条带。

关键词: 芍药, 种子, 休眠, cDNA-AFLP

Abstract: 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 cDNA was digested completely with EcoRⅠand 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 5 μL were taken as a template for selective amplification. About 3 700 differential bands were visualized between before and after breaking hypocotyl dormancy and about 1 600 differential bands were visualized between before and after breaking epicotyl dormancy.

Key words: Paeonia lactiflora, seed, dormancy, cDNA-AFLP

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