摘要： 以蓖麻种子为研究对象，优化其cDNA-AFLP反应体系，为研究不同蓖麻材料或不同发育阶段的蓖麻种子内脂肪酸基因表达差异奠定理论基础。试验结果表明：提取蓖麻种子总RNA；将RNA逆转录为cDNA，用高频限制性内切酶 Eco R Ⅰ和 Mse Ⅰ进行酶切，并与 Eco R Ⅰ、Mse Ⅰ接头连接，得到连接产物；连接产物稀释40倍作为预扩增模板，进行预扩增；优化选择性扩增反应体系，得到最佳反应体系为：10×PCR Buffer 2.0 μL、dNTPs(10 mmol/L)1.4 μL、Mg²⁺(25 mmol/L)1.4 μL、模板稀释80倍1.0 μL、E_x扩增引物(50 pmol/μL) 1.2 μL、M_y扩增引物(50 pmol/μL) 1.2 μL、LA Taq(5 U/μL)0.2 μL、ddH₂O 11.6 μL。采用优化后的体系，利用聚丙烯酰胺凝胶电泳对256对选择性引物进行筛选，得到适合蓖麻种子cDNA-AFLP分析的选择性扩增引物10对。

关键词: 蓖麻, 脂肪酸, cDNA-AFLP

Abstract: Castor is one of the most important oil crops with high economic value.In this work, the cDNA-AFLP system of castor seed was established and optimized to lay a foundation of research fatty acid gene expression in different castor materials or different developmental stages.The main results were as follows:total RNA was extracted and then reversed transcription into cDNA.cDNA digested with EcoR Ⅰ and Mse Ⅰ was joined with EcoR Ⅰ and Mse Ⅰ joint.The jointing products diluted by ddH₂O according to the ratio of 1∶ 40 were used in pre-amplification.And the optimized selective amplification system was performed with 10×PCR Buffer 2.0 μL、dNTPs(10 mmol/L) 1.4 μL、Mg²⁺ (25 mmol/L) 1.4 μL, jointing product 1.0 μL, E_x primer(50 pmol/μL) 1.2 μL, M_y primer(50 pmol/μL) 1.2 μL, LA Taq(5 U/μL) 0.2 μL、ddH₂O 11.6 μL.With the optimized PCR reaction system, we estimated 256 primer combinations with SDS-PAGE and gained 10 combinations which were suitable for cDNA-AFLP analysis of castor seed.

Key words: Castor, Fatty acid, cDNA-AFLP

中图分类号: 

• S565.6