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:40 ng/μI模板DNA 1.0μL、2.5 mmol/L dTPs 2.0μL、5 U/μL TaqNA聚合酶0.2μL、10 μmol/L引物1.0μL、10×PCR Buffer 2.5μL(含有Mg2+),加ddH2O至25.0 μL.PCR反应程序为:94℃预变性5 min;94℃变性40 s,52℃和53℃退火40 s,72℃延伸90s,40个循环;最后72℃延伸7 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大果油茶遗传变异和多样性.
Abstract:
[Objective]The present study was undertaken to establish an ISSR-PCR 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 μμL 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 ISSRPCR amplification reaction system may be useful to assess the genetic variability and diversity in Camellia crepnelliana Tutch.
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