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| 大果油茶基因组DNA提取及ISSR反应体系建立 | |
| 引用本文： | 杨翠芳,陈伯伦,黄诚梅,吕维莉.大果油茶基因组DNA提取及ISSR反应体系建立[J].广西农业科学,2011,42(3):233-235. |
| 作者姓名： | 杨翠芳 陈伯伦 黄诚梅 吕维莉 |
| 作者单位： | 1. 广西农业科学院,南宁,530007;广西作物遗传改良生物技术重点开放实验室,南宁,530007;广西师范大学,广西,桂林,541004 2. 广西农业科学院,南宁,530007 3. 广西农业科学院,南宁,530007;广西作物遗传改良生物技术重点开放实验室,南宁,530007 |
| 基金项目： | 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。 |
| 关 键 词： | 大果油茶 基因组DNA 提取 ISSR反应体系 建立 |
Isolation of genomic DNA and establishment of ISSR reaction system for Camellia crepnelliana Tutch |
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| YANG Cui-fang,CHEN Bo-lun,HUANG Cheng-mei,Lü Wei-li.Isolation of genomic DNA and establishment of ISSR reaction system for Camellia crepnelliana Tutch[J].Guangxi Agricultural Sciences,2011,42(3):233-235. | |
| Authors: | YANG Cui-fang CHEN Bo-lun HUANG Cheng-mei Lü Wei-li |
| Institution: | YANG Cui-fang,CHEN Bo-lun,HUANG Cheng-mei,Lü Wei-li (1 Guangxi Academy of Agricultural Sciences, Nanning 530007, China; 2 Guangxi Crop Genetic Improvement and Biotechnology Lab, Nanning 530007, China; 3 Guangxi Normal University, Guilin, Guangxi 541004, China) |
| 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. |
| Keywords: | Camellia crepnelliana Tutch genomic DNA isolation ISSR reaction system genetic diversity |
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