毛冬青基因组DNA提取方法及ISSR-PCR反应体系的优化  

Optimization of Genomic DNA Extract Method and ISSR-PCR Reaction System for Ilex pubescens

作　　者：付铨盛[1] 卫滢 席秀丽[1] 黄海波[1] 

机构地区：[1]广州中医药大学中药学院,广东广州510006

出　　处：《中国现代中药》2016年第4期415-419,共5页Modern Chinese Medicine

基　　金：国家科技支撑计划(2012BAI29B09);公益性行业科研专项(201407002)

摘　　要：目的:优化毛冬青基因组DNA提取方法,建立稳定性高、重现性好、适合其遗传差异分析的ISSRPCR反应体系。方法:对比CTAB法、mCTAB法、SDS法、蛋白酶K法及植物基因组提取试剂盒的提取效果后,选择mCTAB法结合蛋白酶K法提取毛冬青基因组DNA,并优化了蛋白酶K用量。利用正交试验设计,对毛冬青ISSR-PCR反应的5个因素(Mg^(2+)、dNTPs、Primer、DNA、Taq酶)进行优化,PCR结果使用SPSS16.0软件进行分析,基于优化后体系对哥伦比亚大学公布的100条ISSR引物进行筛选,确定退火温度。结果:采用蛋白酶K-mCTAB法实现了4 h内提取高纯度毛冬青基因组DNA,蛋白酶K用量为20 ng·mL^(-1)时DNA平均提取量较传统CTAB法提升9.5倍;毛冬青ISSR-PCR最终反应体系为Mg^(2+)2.5 mmol·L^(-1),dNTPs 0.15 mmol·L^(-1),Primer 0.4μmol·L^(-1),DNA 70 ng,Taq酶0.5 U。实验从100条引物中筛选出16条引物并确定退火温度,绘制出毛冬青ISSR指纹图谱。结论:蛋白酶K-mCTAB法适合毛冬青基因组DNA的提取,筛选出的反应体系、引物及退火温度能够满足毛冬青ISSR-PCR实验的要求。Objective：To optimize genomic DNA extract method for Ilex pubescens,and establish a stable,reproducible and suitable reaction system for its ISSR analysis of genetic differences.Methods：In this study,five frequently-used methods for plant genomic DNA extraction include CTAB,mCTAB,SDS,protein K and genomic DNA kit were compared for acquiring better DNA product quality and quantity.The optimized method combined mCTAB with Protein K,and the dosage of protein K was also optimized.The ISSR-PCR amplification system of five factors（Mg2＋,dNTPs,primers,Taq DNA polymerase,and DNA template）for I.pubescens was optimized by orthogonal design,the PCR result was analyzed by SPSS 16.0.Then based on the optimal ISSR-PCR amplification system,100 primers were selected and the annealing temperature was proposed by gradient determination.Results：With protein K-mCTAB,the genomic DNA extraction from I.pubescens could be extracted within 4 hours with great quality,and the quantity of extracted DNA was promoted seven times compared with CTAB when Protein K dosage was 20 ng·mL-1.The optimized ISSR-PCR reaction system（20 μL）was constructed,which dosages of Mg2＋,dNTPs,primer,Taq polymerase,and DNA template were 2.5 mmol·L-1,0.15 mmol·L-1,0.4 μmol·L-1,0.5 U,and 70 ng,respectively.16 primers was selected and its annealing temperature was proposed.Conclusion：Proteinase K-mCTAB is suitable for genomic DNA extraction of I.pubescens,the reaction system,primers and its annealing temperature is stable,reproducible,and suitable for its ISSR-PCR analysis

关 键 词：毛冬青 DNA提取 反应体系优化 引物筛选 

分 类 号：R284.2[医药卫生—中药学]