秦岭野生蕙兰RAPD反应体系的优化  

Optimization of RAPD reaction system on wild Cymbidium faberiin Qinling Mountain

作　　者：王宝宁[1] 张显[2] 陈君梅[1] 宋军阳[1] 

机构地区：[1]西北农林科技大学林学院,陕西杨凌712100 [2]西北农林科技大学园艺学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2011年第12期87-94,共8页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家林业局社会公益项目(200704009);陕西省农业攻关项目(2009K01-11);西安市科技局攻关项目(NC09045-1)

摘　　要：【目的】建立适宜于秦岭野生蕙兰RAPD分析的PCR反应条件,为利用RAPD技术研究秦岭野生蕙兰的遗传多样性提供技术依据。【方法】以秦岭野生蕙兰叶片为材料,采用改良CTAB法,提取野生蕙兰基因组DNA进行RAPD扩增反应。以5′-CCTTGACGCA-3′(S12)为随机引物,通过L16(45)正交试验与单因素试验2种方法,对RAPD反应体系的主要参数(Mg2+浓度、dNTPs浓度、模板DNA用量、引物浓度、Taq DNA聚合酶用量)进行摸索和优化,并经过验证试验,建立适合秦岭野生蕙兰的RAPD遗传多样性分析体系。【结果】研究得到的适于秦岭野生蕙兰的RAPD遗传多样性分析体系为:25μL反应体系中,Mg2+浓度为2.5μmol/L,dNTPs浓度为0.16mmol/L,模板DNA用量为100ng,引物浓度0.6μmol/L,Taq DNA聚合酶用量为1.5U。各因素对RAPD反应的影响程度依次为:模板DNA用量>引物浓度>Taq DNA聚合酶用量>dNTPs浓度>Mg2+浓度。【结论】优化的秦岭野生蕙兰RAPD反应体系扩增效果比较理想,扩增条带的多态性明显、清晰度高、重复性好,可用于进一步的遗传多样性分析。【Objective】 The reaction system RAPD of wild Cymbidium faberi in Qinling was established to provide technical basis of technology research for the genetic diversity wild Qinling orchids.【Method】 In this research,modified CTAB method was used to extract DNA from the leaves of C.faberi Rolfe for RAPD reaction.Primer 5′-CCTTGACGCA-3′（S12） was used as random primer,L16（45） orthogonal design and single factor design were used to screen and to establish optimal reaction system of RAPD that were used in wild C.faberi in Qinling.Mg2＋,dNTPs,primer,Taq DNA polymerase,and template DNA were used as important parameters for exploration and optimization experiment.【Result】 The optimum concentration of five important components such as Mg2＋,Taq DNA polymerase,dNTPs,primer,and template DNA in 25 μL reaction system was 2.5 μmol/L,1.5 U,0.16 mmol/L,0.6 μmol/L and 100 ng.The effective degree of the factors on the RAPD reaction was in the order：template DNAprimerTaq DNA polymerasedNTPsMg2＋.【Conclusion】 The amplification effect of the optimization RAPD reaction system of wild C.faberi in Qinling is ideal.Amplified polymorphism of strip is obvious,reproducible and with high definition,which can be used for further analysis of genetic diversity.

关 键 词：秦岭 野生蕙兰 RAPD-PCR 体系优化 

分 类 号：S682.310.1[农业科学—观赏园艺]