蓖麻种子cDNA-AFLP反应体系的优化  

Optimization of the cDNA-AFLP System of Castor Seed

作　　者：陈晓凤[1,2] 黄凤兰[1,2] 李国瑞[1,2] 王文跃[1,2] 张智勇[3,4] 包春光 吴春桃[1] 张现世 

机构地区：[1]内蒙古民族大学生命科学学院,内蒙古通辽028000 [2]内蒙古高校蓖麻产业工程技术研究中心,内蒙古通辽028000 [3]内蒙古民族大学农学院,内蒙古通辽028000 [4]内蒙古通辽市农业科学研究院,内蒙古通辽028015

出　　处：《华北农学报》2014年第3期74-80,共7页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金项目(30760123;31160290;31060194);内蒙古自然科学基金项目(2010BS0511);内蒙古人才基金项目;内蒙古民族大学市校合作项目(SXZD2012018);内蒙古民族大学教育教学研究项目(No.2009053)

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EcoRⅠ和MseⅠ进行酶切,并与EcoRⅠ、MseⅠ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 2.0μL、dNTPs(10 mmol/L)1.4μL、Mg2+(25 mmol/L)1.4μL、模板稀释80倍1.0μL、Ex扩增引物(50 pmol/μL)1.2μL、My扩增引物(50 pmol/μL)1.2μL、LA Taq(5 U/μL)0.2μL、ddH2O 11.6μL。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。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,Exprimer（ 50 pmol /μL） 1.2 μL,Myprimer（ 50pmol /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.

关 键 词：蓖麻 脂肪酸 CDNA-AFLP 

分 类 号：S565.6[农业科学—作物学]