石斛属植物ISSR扩增体系的建立与优化
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摘要
旨在建立并优化石斛属植物的ISSR-PCR反应体系。通过对ISSR-PCR反应体系中主要影响因子模板DNA、dNTP、10×PCR Buffer、引物以及Taq DNA聚合酶分别进行单因素优化,最终建立一套适用于石斛属植物的扩增多态性高、稳定性强、带型清晰的ISSR最佳反应体系。20μL反应体系的适宜浓度及用量分别为:模板DNA 10 ng/μL 3μL,dNTP 2.5 mmol/L 1.5μL,10×PCR Buffer 3.0μL,引物4μmol/L3.5μL,TaqDNA聚合酶5 U 0.2μL。这一优化体系适用于石斛ISSR分析,为今后遗传多样性与亲缘关系分析、连锁图谱构建、QTL定位、基因定位与克隆等方面的研究提供了技术支撑。
The research aimed to establish and optimize a stable ISSR-PCR reaction system for Dendrobium.In this paper, the key factors of ISSR-PCR reaction system including DNA template concentration, dNTP, 10×PCR Buffer, primer concentration, DNA Taq polymorphism were investigated and optimized. The stablereaction system could amplify high levels of polymorphism, good repeatability and clear band patterns. ISSRsystem(total volume of 20 μL) established was as follows: template DNA 10 ng/μL 3 μL, dNTP 2.5 mmol/L1.5 μL, 10×PCR Buffer 3.0 μL, primer 4 μmol/L 3.5 μL, Taq polymorphism 5 U 0.2 μL. It showed that theoptimized system in this experiment could be applied in the ISSR analysis for Dendrobium. This researchprovided technical support for genetic diversity and genetic relationship research, linkage map construction,QTL location, gene mapping and gene cloning and so on.
The research aimed to establish and optimize a stable ISSR-PCR reaction system for Dendrobium.In this paper, the key factors of ISSR-PCR reaction system including DNA template concentration, dNTP, 10×PCR Buffer, primer concentration, DNA Taq polymorphism were investigated and optimized. The stablereaction system could amplify high levels of polymorphism, good repeatability and clear band patterns. ISSRsystem(total volume of 20 μL) established was as follows: template DNA 10 ng/μL 3 μL, dNTP 2.5 mmol/L1.5 μL, 10×PCR Buffer 3.0 μL, primer 4 μmol/L 3.5 μL, Taq polymorphism 5 U 0.2 μL. It showed that theoptimized system in this experiment could be applied in the ISSR analysis for Dendrobium. This researchprovided technical support for genetic diversity and genetic relationship research, linkage map construction,QTL location, gene mapping and gene cloning and so on.
引文
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[2]Gupta M,Chyi Y S,Romero-Severson J,et al.Amplification of DNA markers from evolutionarily diverse gemomes using single primers of simple-sequence repeats[J].Theoretical and Applied Genetics,1994,89:998-1006.
[3]Ratnaparkhe B,Tekeoglu M,Muehlbauer F J.Inter simple sequence repeat(ISSR)polymorphisms are useful for finding markers associated with disease resistance gene clusters[J].Theoretical and Applied Genetics,1998,97:515-519.
[4]Okpul T,Mace E S,Godwin I D,et al.Evaluation of variability among breeding lines and cultivars of Taro(Colocasia Esculenta)in papua new guinea using ISSR fingerprinting and agromorphological characterization[J].Plant Genetic Resources Newsleter,2005,143:8-16.
[5]赵杨,陈晓阳,王秀荣,等.9种胡枝子亲缘关系的ISSR分析[J].吉林林业科技,2006,35(2):1-4.
[6]梁景霞,祁建民,方平平,等.烟草种质资源遗传多样性与亲缘关系的ISSR聚类分析[J].中国农业科学,2008,41(1):286-294.
[7]Joseph S,David W R.黄培堂等译.分子克隆实验指南[M].北京:化学工业出版社,2008:385-396.
[8]魏丹红,徐红,王燕燕,等.金钗石斛遗传多样性研究的ISSR-PCR反应体系建立与优化[J].上海中医药大学学报,2007,21(4):64-68.
[9]武荣花.我国石斛属植物种质资源及其亲缘关系研究[D].北京:中国林业科学研究院,2007.
[10]杨立昌,邓辉,乙引,等.药用石斛ISSR分子标记研究[J].中药材,2010,33(12):1841-1844.
[11]杨春勇,李学兰,王云强,等.人工栽培石斛的ISSR标记分析[J].中国农学通报,2011,27(4):148-152.
[12]石锐,郭长虹.聚丙烯酰胺凝胶中DNA的银染方法[M].生物技术,1998,8(6):46-48.
[13]吴冠芸,潘华珍,吴羽.生物化学与分子生物学实验常用数据手册[M].北京:科学出版社,1999.
[14]林万明.PCR技术操作和应用指南[M].北京:人民军医出版社,1993,7-14.
[15]李廷春,高正良,汤志顺.丹参SRAP反应体系的建立与优化[J].生物学杂志,2008,25(1):40-44.
[16]夏志强,邹枚伶,王文泉.木薯SRAP扩增体系的建立与优化[J].中国农学通报,2008,24(9):457-460.
[17]申洁,侯思宇,孙朝霞,等.正交优化枣树ISSR-PCR反应体系的研究[J].华北农学报,2010,25(2):116-120.
[18]邱丽娜,廖明安,李明章,等.正交设计对红阳弥胡桃ISSR反应体系的优化研究[J].北方园艺,2010,(16):125-128.
[19]宁静,黄建安,李娟,等.茶树ISSR-PCR反应体系的正交优化[J].湖南农业大学学报:自然科学版,2010,36(4):414-417.
[20]胡薇,黄儒珠,孙端,等.均匀设计优化建兰ISSR-PCR体系[J].生命科学研究,2007,11(1):58-63.
[2]Gupta M,Chyi Y S,Romero-Severson J,et al.Amplification of DNA markers from evolutionarily diverse gemomes using single primers of simple-sequence repeats[J].Theoretical and Applied Genetics,1994,89:998-1006.
[3]Ratnaparkhe B,Tekeoglu M,Muehlbauer F J.Inter simple sequence repeat(ISSR)polymorphisms are useful for finding markers associated with disease resistance gene clusters[J].Theoretical and Applied Genetics,1998,97:515-519.
[4]Okpul T,Mace E S,Godwin I D,et al.Evaluation of variability among breeding lines and cultivars of Taro(Colocasia Esculenta)in papua new guinea using ISSR fingerprinting and agromorphological characterization[J].Plant Genetic Resources Newsleter,2005,143:8-16.
[5]赵杨,陈晓阳,王秀荣,等.9种胡枝子亲缘关系的ISSR分析[J].吉林林业科技,2006,35(2):1-4.
[6]梁景霞,祁建民,方平平,等.烟草种质资源遗传多样性与亲缘关系的ISSR聚类分析[J].中国农业科学,2008,41(1):286-294.
[7]Joseph S,David W R.黄培堂等译.分子克隆实验指南[M].北京:化学工业出版社,2008:385-396.
[8]魏丹红,徐红,王燕燕,等.金钗石斛遗传多样性研究的ISSR-PCR反应体系建立与优化[J].上海中医药大学学报,2007,21(4):64-68.
[9]武荣花.我国石斛属植物种质资源及其亲缘关系研究[D].北京:中国林业科学研究院,2007.
[10]杨立昌,邓辉,乙引,等.药用石斛ISSR分子标记研究[J].中药材,2010,33(12):1841-1844.
[11]杨春勇,李学兰,王云强,等.人工栽培石斛的ISSR标记分析[J].中国农学通报,2011,27(4):148-152.
[12]石锐,郭长虹.聚丙烯酰胺凝胶中DNA的银染方法[M].生物技术,1998,8(6):46-48.
[13]吴冠芸,潘华珍,吴羽.生物化学与分子生物学实验常用数据手册[M].北京:科学出版社,1999.
[14]林万明.PCR技术操作和应用指南[M].北京:人民军医出版社,1993,7-14.
[15]李廷春,高正良,汤志顺.丹参SRAP反应体系的建立与优化[J].生物学杂志,2008,25(1):40-44.
[16]夏志强,邹枚伶,王文泉.木薯SRAP扩增体系的建立与优化[J].中国农学通报,2008,24(9):457-460.
[17]申洁,侯思宇,孙朝霞,等.正交优化枣树ISSR-PCR反应体系的研究[J].华北农学报,2010,25(2):116-120.
[18]邱丽娜,廖明安,李明章,等.正交设计对红阳弥胡桃ISSR反应体系的优化研究[J].北方园艺,2010,(16):125-128.
[19]宁静,黄建安,李娟,等.茶树ISSR-PCR反应体系的正交优化[J].湖南农业大学学报:自然科学版,2010,36(4):414-417.
[20]胡薇,黄儒珠,孙端,等.均匀设计优化建兰ISSR-PCR体系[J].生命科学研究,2007,11(1):58-63.