大果油茶基因组DNA提取及ISSR反应体系建立(英文)  

Isolation of genomic DNA and establishment of ISSR reaction system for Camellia crepnelliana Tutch

作　　者：杨翠芳[1,2,3] 陈伯伦[1] 黄诚梅[1,2] 吕维莉[1,2] 

机构地区：[1]广西农业科学院,南宁530007 [2]广西作物遗传改良生物技术重点开放实验室,南宁530007 [3]广西师范大学,广西桂林541004

出　　处：《南方农业学报》2011年第3期233-235,共3页Journal of Southern Agriculture

基　　金：Guangxi Natural Science Foundation Item(2010GXNSFA0138081)

摘　　要：【目的】建立大果油茶的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】以大果油茶嫩叶片为供试材料,采用改良的SDS法提取其基因组DNA,并对其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,OD260/OD280值在1.8～2.0之间,DNA无降解现象,完全可以满足ISSR-PCR扩增要求。在25.0μLISSR-PCR反应体系中,各组分的适宜浓度配比为:40ng/μL模板DNA1.0μL、2.5mmol/LdNTPs2.0μL、5U/μLTaqDNA聚合酶0.2μL、10μmol/L引物1.0μL、10×PCR Buffer2.5μL(含有Mg2+),加ddH2O至25.0μL。PCR反应程序为:94℃预变性5min;94℃变性40s,52℃和53℃退火40s,72℃延伸90s,40个循环;最后72℃延伸7min。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大果油茶遗传变异和多样性。【Objective】The present study was undertaken to establish an ISSRPCR amplification reaction system for Camellia crepnelliana Tutch for providing basis to further research. 【Method】The young leaves of Camellia crepnelliana Tutch were used to isolate high-quality genomic DNA using the modified SDS method. The different conditions for ISSR reaction system were optimized. 【Result】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 components of the best ISSR-PCR amplification system in a total 25.0 μL reaction volume included 1.0 μL of template DNA （40 ng/μL）,2.0 μL of dNTPs （2.5 mmol/L）,0.2 μL of Taq DNA polymerase （5 U/μL）,1.0 μL primer （10 μmol/L）, 2.5 μL 10×PCR buffer（plus Mg2＋）. The PCR program was set to 5 min at 94℃ for pre-denaturing,followed by 40 cycles of 40 s at 94℃（denaturation）,40 s at 52℃ and 53℃（annealing）and 90 s at 72℃（extension）,the final extension was set at 72℃ for 7 min. The optimized reaction system gave distinct DNA fingerprinting results. 【Conclusion】The established ISSR-PCR amplification reaction system may be useful to assess the genetic variability and diversity in Camellia crepnelliana Tutch.

关 键 词：大果油茶 基因组DNA 提取 ISSR反应体系 建立 

分 类 号：S794.401[农业科学—林木遗传育种]