珠子参ISSR-PCR反应体系的建立及优化  

Establishment and optimization of ISSR-PCR reaction system for Panax japonicus C.A.Mey.var.major

作　　者：宋小妹[1] 许苗苗[1] 刘银环[1] 王薇[1] 刘超[1] 杨新杰[1] 崔九成[1] 

机构地区：[1]陕西中医学院,咸阳712046

出　　处：《西北药学杂志》2014年第6期551-554,共4页Northwest Pharmaceutical Journal

基　　金：国家自然科学基金项目(编号:81102805/H2804);陕西省科技统筹创新工程项目(编号:2011KTCQ03-02);陕西省科学技术研究发展计划项目(编号:2011K16-02-02);陕西省教育厅项目(2010JK502);陕西省中医管理局专项科研计划项目(编号:ZY08)

摘　　要：目的：建立珠子参的ISSR-PCR适宜扩增反应体系和程序，为其深入研究奠定基础。方法采用新型植物基因组DNA提取试剂盒提取珠子参总DNA ，并用正交设计实验对其ISSR反应体系条件进行筛选及优化。结果提取的基因组DNA纯度和完整性较好，A260/A280值在1．8～2．0之间，DNA无降解现象，可满足ISSR-PCR扩增要求。珠子参ISSR分析的最适反应体系为：在20μL PCR反应体积中，含10 ng模板DNA、0．20 mmol·L -1 dNTPs、1．5μmol·L -1引物、3U TaqDNA 聚合酶、2μL 10× PCR Buffer、3 mmol·L -1 Mg2＋。扩增程序为：95℃预变性5 min ，94℃变性30s，58℃退火30 min ，72℃延伸1 min ，循环40次，72℃延伸10 min。由此可获得带型丰富和清晰可辨的DNA指纹图谱。结论建立的ISSR-PCR反应体系可用于研究珠子参的遗传变异和遗传多样性。Objective The present study was undertaken to establish an ISSR-PCR amplification reaction system for Panax japoni-cus C .A .Mey .var .major for providing basis to further research .Methods The samples of Panax japonicus C .A .Mey .var . major were used to isolate high-quality genomic DNA by using the plant genomic DNA Preps Kit .The different conditions for IS-SR reaction system were optimized .Results The extracted genomic DNA was of high quality as revealed by 1 .8 to 2 .0 absorbance ratio of wavelengths （260/280） ,did not show any degradation ,and was found suitable for ISSR-PCR reaction .The conditions of the optimal ISSR-PCR amplification system were in a total 20μL reaction volume included 10 ng of template DNA ,0 .20 mmol · L-1 of dNTPs ,1 .5 μmol · L -1 primer ,3 U of Taq DNA polymerase ,2 μL 10 × PCR buffer and 3 mmol · L -1 Mg2＋ .The PCR program was set to 5 min at 95 ℃ for pre-denaturing ,followed by 40 cycles of 30 s at 94 ℃ （denaturation） ,30 s at 58 ℃ （annealing） and 1 min at 72 ℃ （extension） ,and the final extension was set at 72 ℃ for 10 min .The optimized reaction system yielded distinct DNA fingerprinting results .Conclusion The established ISSR-PCR amplification reaction system may be useful to assess the genetic variability and diversity in Panax j aponicus C .A .Mey .var .major .

关 键 词：珠子参 基因组DNA ISSR反应体系 遗传多样性 

分 类 号：R282[医药卫生—中药学]