春兰基因组DNA提取及RAPD反应体系的优化

摘要/Abstract

摘要： 以春兰为研究材料,对春兰基因组DNA的提取以及RAPD-PCR反应体系进行了优化.结果表明,在提取液中加入PVP和β-巯基乙醇去除酚类物质,采用高盐沉淀DNA和多次洗涤去除糖类,可以得到高质量春兰基因组DNA.电泳检测表明,所获得的DNA完整,无降解,完全可以满足RAPD等分子标记分析的需要.同时对春兰RAPD-PCR反应体系中各个影响因素进行了优化,建立了适合春兰RAPD分子标记的最佳反应体系,即20μl反应体系中含1UTaqDNA聚合酶,60μmol/L dNTPs,2.25mmol/L Mg2+,1.0μmol/L引物,1.5ng/μl模板DNA.

关键词: 西瓜, 西瓜, 嫁接砧木, 抗病性, 经济性状

中图分类号:

S663.1

单丽丽　陆瑞菊　王亦菲　陆惠丽　黄剑华. 春兰基因组DNA提取及RAPD反应体系的优化[J]. 中国农学通报, 2008, 24(1): 68-73.

Shan Lili　 Lu Ruiju　 Wang Yifei　 Lu Huili　 Huang Jianhua. Extraction of Genomic DNA and Optimization of RAPD Reaction System of Cymbidium goeringii[J]. Chinese Agricultural Science Bulletin, 2008, 24(1): 68-73.

参考文献

[1]罗毅波.国兰野生资源保护及国兰产业发展.濒危物种科学通讯,2005,14(1).
[2]陈惠云,孙志栋,茅轶俊,等.春兰基因组DNA提取方法的研究.分子植物育种,2006,4(1):135-142.
[3]Baudracco-Amas S,Pitrat M.A genetic map of melon (Cucumis melo L.) with RFLP、RAPD markers.Theor Appl Genet,1996,93:57-64.
[4]Wang Y H,Thomas C E,Dan R A.A genetic map of melon (Cucumis melo L.) based on amplified fragment length polymorphism(AFLP) markers.Thero Appl Genet,1997,95:791-789.
[5]Park Y,Katzir N,Brotman Y,et al.Comparative mapping of ZYMV resistances in cucumber (Cucumis sativus L.) and melon (Cucumis melo L.).Thero Appl Genet,2004,109(4):707-712.
[6]Wang Y H,Thomas C E,Dean R A.Genetic mapping ofa fusarium wilt resistance gene (Fom-2) in melon (Cucumis melo L.).Molecular Breeding,2000,6(4):379-389.
[7]Karsies T,Dean R,Thomas C.Toward the development of molecular markers linked to race 2 Fusarium wilt resistance in melon (Cucumis melo L.).Acta Hort,2000,510:415-419.
[8]Brotman Y,Silberstein I,Kovalski,et al.Lingage groups of Cucumis melo.L including resistance gene homologues and known genes.Acta Hort,2000,510:441-448.
[9]Williams J G K,Kubelik A R,Livak K J,et al.DNA Polymerphisms amplified by arbimary primers are useful as genetic markers.Nucleic Acids Res,1990,18:6531-6534.
[10]Welsh J,McClelland M.Finger printing genomes using PCR with arbitrary primers.Nucleic Acids Res,1990,18:7213-7218.
[11]王同坤,董超华,马艳,等.板栗SSR和RAPD技术体系的建立.果树学报,2006,23(6):825-829.
[12]李庆云,庄东红,胡忠,等.花生不同部位对提取DNA的影响.花生学报,2003,32(2):16-20.
[13]高丽,杨波.春兰ISSR-PCR反应体系的优化.华中农业大学学报,2006,25(3):305-309.
[14]MacPherson J M,Eckstein P E,Scoles G J,et al.Variability of the random amplified polymorphic DNA assay among thermal cyclers and effects of primer and DNA concentration.Molecular and Cellular Probes,1993,7(2):289-293.
[15]Jones C J,Edwards K J,Castaglione S,et al.Repro-ducibility testing of RAPD,AFLP and SSR markers in plants by a network of European laboratories.Molecular Breeding,1997,3(5):381-390.
[16]Lowe A J,Hanotte O,Guarino L.Standardization of molecular genetic techniques for the characterization of germplasm collections:the case of random amplified polymorphic DNA (RAPD).Plant Genet Resource News,1996,107(1):50-54.
[17]Rothuizen J,Wolferen M.Randomly amplified DNA polymorphisms in dogs are reproduceible and display Mendelian transmission.Animal Genetics,1994,25(1):13-18.
[18]汪小全,邹喻苹,张大明,等.RAPD应用于遗传多样性和系统学研究中的问题.植物学报,1996,38(12):954-962.
[19]张青林,罗正荣.柿属植物ISSR分析技术的建立.农业生物技术学报,2004,12(5):521-525.
[20]张海英,王永健,许勇,等.黄瓜种质资源遗传亲源关系的RAPD分析.园艺学报,1998,25(4):345-349.