野百合AFLP反应体系的建立及优化  

Establishment and Optimization of an AFLP Reaction System for Lilium brownii

作　　者：柯贤锋[1] 智丽[1] 眭顺照[2] 秦华[2] 李名扬[2] 李先源[2] 

机构地区：[1]西南大学园艺园林学院,重庆400716 [2]西南大学花卉研究所,重庆400716

出　　处：《西南大学学报（自然科学版）》2007年第12期100-103,共4页Journal of Southwest University(Natural Science Edition)

基　　金："十一五"国家科技基础条件平台建设专项资助项目(2005DKA21403);重庆市自然基金资助项目(2005BB1129);重庆市教委资助项目(KJ050209).

摘　　要：以重庆市巫山和巫溪两地的野百合为实验材料,对实验中涉及的各个重要试剂,如接头浓度、预扩和选扩模板稀释倍数及dNTP等的用量进行梯度比较,确定最佳用量.结果表明:①改良的CTAB法提取的野百合基因组DNA经过纯化后符合AFLP实验要求;②接头浓度为50pmol/μL较好;③酶切连接产物稀释20倍,dNTP用量为1.0μL(50μL体系),72℃开始扩增较好;④预扩增产物稀释30倍最佳.⑤从64个引物组合中筛选出符合野百合AFLP反应的20对引物组合.因此,优化后的体系适合野百合遗传多样性研究.Wild Lilium brownii plants collected from Wushan and Wuxi were used as experiment materials. Various important influencing factors （and their dosages） were compared, including the methods of genomic DNA extraction, adaptor concentrations, template concentrations and dNTP, and the optimized combination was identified. The results indicated： （1） the L. brozvnii genomic DNA extracted by a modified CTAB procedure was fit for AFLP experiment after purification; （2） the best adaptor concentration was 50 pmol/μL; （2） the ligation products should be diluted 20 folds, 1.0μL dNTP should be mixed in 50μL PCR reaction solution, and the pre-amplification reaction should begin at 72 ℃ instead of at 94 ℃. （4） the preamplification products should be diluted 30 folds. （5） Twenty pairs of primer combinations were screened from 64 EcoR1/Msel primer combinations. The optimized AFLP reaction system was fit for the research of the genetic diversity of L. brovonie.

关 键 词：野百合 AFLP 体系优化 

分 类 号：Q75[生物学—分子生物学] TQ323.8[化学工程—合成树脂塑料工业]