泽泻ISSR反应体系的建立与优化
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摘要
目的:建立并优化泽泻反应体系和扩增程序,筛选出多态性、重复性较好的ISSR引物,为泽泻种源鉴别以及指纹图谱的构建提供理论基础。方法:利用单因素试验法,对Mg~(2+)浓度、dNTPs浓度、TaqDNA聚合酶浓度、引物浓度及模板DNA含量这5个PCR反应体系主要成分以及退火温度进行筛选,并利用建立优化的反应体系和扩增程序对以往泽泻文献报道的ISSR引物进行筛选。结果:建立了最佳PCR反应体系:20μL的反应液中含2.0mmol·L~(-1)Mg~(2+),0.4mmol·L~(-1)d NTPs,1.0 U Taq DNA聚合酶,0.4mol·L~(-1)引物,10ng模板DNA,2μL10x Buffer,13.1μL dd H_2O。反应程序为:94℃预变性5min;94℃变性45s。58.9℃退火45s,72℃延伸90s,40个循环;72℃延伸7min,4℃保存。结论:利用优化的泽泻的反应体系,筛选出15条多态性较好的引物,并对泽泻的21个种源进行ISSR扩增,扩增出的条带清晰、多态性较高,证实了该体系具有较高稳定性、重现性和适用性。此体系也为泽泻种源间的遗传多样性研究以及ISSR分子标记奠定了基础。
Objectives:To establish optimized ISSR system of Alisma orientale(Samuel)Juz for selection of ISSR prim-ers with high stability and repeatability. Methods:Applied the single factor design for optimizing five factors on ISSRamplification,such as Mg2+concentration,d NTPs concentration,Taq DNA polymerase concentration,primer concen-tration and template DNA concentration.Results:The results showed that the optimal 20μL ISSR-PCR reaction sys-tem for Alisma orientale(Samuel)Juz contained 2.0mmol/L Mg2 +,0.4mmol/L d NTPs,1.0 U Taq DNA polymerase,0.4mol/L primer,10 ng DNA template,2μL 10 x Buffer,13.1 μL dd H2 O.On this optimal ISSR amplification system,15 primers were screened with good polymorphism baased on previous achievement on Alisma orientale(Samuel)Juzprimers.Conclusion:15 primers with high polymorphism were effectively selected based on the optimized ISSR sys-tem.The test showed that the system was stable,reliable and applicative. The optimal ISSR-PCR reaction system issuitable for the study of genetic diversity in plant of Alisma orientale(Samuel)Juz in different provenances.
Objectives:To establish optimized ISSR system of Alisma orientale(Samuel)Juz for selection of ISSR prim-ers with high stability and repeatability. Methods:Applied the single factor design for optimizing five factors on ISSRamplification,such as Mg2+concentration,d NTPs concentration,Taq DNA polymerase concentration,primer concen-tration and template DNA concentration.Results:The results showed that the optimal 20μL ISSR-PCR reaction sys-tem for Alisma orientale(Samuel)Juz contained 2.0mmol/L Mg2 +,0.4mmol/L d NTPs,1.0 U Taq DNA polymerase,0.4mol/L primer,10 ng DNA template,2μL 10 x Buffer,13.1 μL dd H2 O.On this optimal ISSR amplification system,15 primers were screened with good polymorphism baased on previous achievement on Alisma orientale(Samuel)Juzprimers.Conclusion:15 primers with high polymorphism were effectively selected based on the optimized ISSR sys-tem.The test showed that the system was stable,reliable and applicative. The optimal ISSR-PCR reaction system issuitable for the study of genetic diversity in plant of Alisma orientale(Samuel)Juz in different provenances.
引文
[1]中国植物志编委会.中国植物志[M].北京:科学出版社,1992:141-142.
[2]Liu Y Q,Chen X F,Yang W Y,et al.Dynamic accumulation of chromium in Alisma orientale and its effect on growth of Alisma orientale under nitrogen-chromium coupling[J].Chinese Traditional&Herbal Drugs,2012,43(5):978-983.
[3]中华人民共和国卫生部药典委员会.中国药典一部[M].北京:化学工业出版社,2005:158.
[4]Wang C,Zhang J X,Shen X L,etal.Reversal of Pglycoproteinmediated multidrug resistance by alisol B23acetate[J].Biochemical Pharmacology,2004:843.
[5]胡珊梅,许瑜瑜.建泽泻、川泽泻及江泽泻HPLC特征图谱比较研究[J].药物分析杂志,2010,03:409-414.
[6]贺佳,丁小余,褚必海,等.泽泻ISSR反应体系的建立与优化[J].南京师大学报(自然科学版),2006,03:86-90.
[7]Vijayan K,Chatterjee S N.ISSR profiling of Indian cultivars of mulberry(Morus,spp.)and its relevance to breeding programs[J].Euphytica,2003,131(1):53-63.
[8]Othman N,Decraene J,Cai W,et al.Establishment and Optimization of ISSR-PCR Reaction System for Clematis fasciculifloora Franch.[J].Journal of Yunnan Agricultural University,2012,3(1):639-643.
[9]汪结明,项艳,吴大强,等.杨树ISSR反应体系的建立及正交设计优化[J].核农学报,2007,21(5):470-473.
[10]Zeng Y L,Xie P,Xie Y J,et al.Establishment and Optimization of Eucalyptus ISSR-PCR Reaction System[J].Journal of Central South University of Forestry&Technology,2008,28(1):44-48.
[11]Yang H Y,Liu C G,Bai X J.Establishment and Optimization of ISSR-PCR Reaction System for Columba domestica[J].China Animal Husbandry&Veterinary Medicine,2012,39(5):44-47.
[12]Ying Y U,Guo J,Wang Z Q,et al.Establishment and optimization of ISSR-PCR reaction system for Platycodon grandiflorus[J].Lishizhen Medicine&Materia Medica Research,2014,25(6):1396-1399.
[13]吴鑫,雷天刚,何永睿,等.柑桔SRAP和ISSR分子标记技术体系的建立与优化[J].分子植物育种,2008,6(1):170-176.
[14]郭凌飞,邹明宏,曾辉,等.澳洲坚果ISSR-PCR反应体系的建立与优化[J].林业科学,2008,05:160-164.
[15]胡甦,王永清,陶炼,等.三角紫叶酢浆草ISSR反应体系的建立与优化[J].草业学报,2011,20(5):142-150.
[16]何正文,刘运生,陈立华,等.正交设计直观分析法优化PCR条件[J].湖南医科大学学报,1998,04:76-77.
[17]Ou L J,Yan W,Liao Y X,et al.Establishment and optimization of ISSR-PCR reaction system in Asparagus cochinchinensis[J].Chinese Traditional&Herbal Drugs,2011,42(2):353-357.
[18]田艳伶,李志辉,杨模华,等.钩栗ISSR-PCR反应体系的建立与优化[J].中南林业科技大学学报,2015(2):32-37.
[19]冯夏莲,何承忠,张志毅,等.毛白杨ISSR反应体系的建立及优化[J].北京林业大学学报,2006,03:61-65.
[2]Liu Y Q,Chen X F,Yang W Y,et al.Dynamic accumulation of chromium in Alisma orientale and its effect on growth of Alisma orientale under nitrogen-chromium coupling[J].Chinese Traditional&Herbal Drugs,2012,43(5):978-983.
[3]中华人民共和国卫生部药典委员会.中国药典一部[M].北京:化学工业出版社,2005:158.
[4]Wang C,Zhang J X,Shen X L,etal.Reversal of Pglycoproteinmediated multidrug resistance by alisol B23acetate[J].Biochemical Pharmacology,2004:843.
[5]胡珊梅,许瑜瑜.建泽泻、川泽泻及江泽泻HPLC特征图谱比较研究[J].药物分析杂志,2010,03:409-414.
[6]贺佳,丁小余,褚必海,等.泽泻ISSR反应体系的建立与优化[J].南京师大学报(自然科学版),2006,03:86-90.
[7]Vijayan K,Chatterjee S N.ISSR profiling of Indian cultivars of mulberry(Morus,spp.)and its relevance to breeding programs[J].Euphytica,2003,131(1):53-63.
[8]Othman N,Decraene J,Cai W,et al.Establishment and Optimization of ISSR-PCR Reaction System for Clematis fasciculifloora Franch.[J].Journal of Yunnan Agricultural University,2012,3(1):639-643.
[9]汪结明,项艳,吴大强,等.杨树ISSR反应体系的建立及正交设计优化[J].核农学报,2007,21(5):470-473.
[10]Zeng Y L,Xie P,Xie Y J,et al.Establishment and Optimization of Eucalyptus ISSR-PCR Reaction System[J].Journal of Central South University of Forestry&Technology,2008,28(1):44-48.
[11]Yang H Y,Liu C G,Bai X J.Establishment and Optimization of ISSR-PCR Reaction System for Columba domestica[J].China Animal Husbandry&Veterinary Medicine,2012,39(5):44-47.
[12]Ying Y U,Guo J,Wang Z Q,et al.Establishment and optimization of ISSR-PCR reaction system for Platycodon grandiflorus[J].Lishizhen Medicine&Materia Medica Research,2014,25(6):1396-1399.
[13]吴鑫,雷天刚,何永睿,等.柑桔SRAP和ISSR分子标记技术体系的建立与优化[J].分子植物育种,2008,6(1):170-176.
[14]郭凌飞,邹明宏,曾辉,等.澳洲坚果ISSR-PCR反应体系的建立与优化[J].林业科学,2008,05:160-164.
[15]胡甦,王永清,陶炼,等.三角紫叶酢浆草ISSR反应体系的建立与优化[J].草业学报,2011,20(5):142-150.
[16]何正文,刘运生,陈立华,等.正交设计直观分析法优化PCR条件[J].湖南医科大学学报,1998,04:76-77.
[17]Ou L J,Yan W,Liao Y X,et al.Establishment and optimization of ISSR-PCR reaction system in Asparagus cochinchinensis[J].Chinese Traditional&Herbal Drugs,2011,42(2):353-357.
[18]田艳伶,李志辉,杨模华,等.钩栗ISSR-PCR反应体系的建立与优化[J].中南林业科技大学学报,2015(2):32-37.
[19]冯夏莲,何承忠,张志毅,等.毛白杨ISSR反应体系的建立及优化[J].北京林业大学学报,2006,03:61-65.