摘要
建立并研究适合红叶石楠的RAPD的最佳反应体系,为以后开展红叶石楠的遗传多样性研究、物种资源研究和亲缘关系鉴定等提供重要的参考。采用改进后的CTAB法,成功地提取了红叶石楠基因组DNA,通过单因素试验设计,研究了Mg2+、dNTP、引物的浓度和模板DNA,Taq酶的用量对RAPD反应的影响,建立适合于红叶石楠的RAPD反应体系。结果表明,在25μL的RAPD反应体系中最佳反应条件分别是10×PCRBuffer2.5μL、Mg2+(25mmol/L)2.0μL、引物(20μmol/L)2.0μL、dNTPs(2.5mmol/L)1.8μL、DNA模板(49.5μg/mL)2.0μL、Taq聚合酶0.3μL(5U/μL)。适宜的PCR循环程序为94℃预变性4min,然后44个循环(94℃变性30s,36℃退火30s,72℃延伸2min)后,72℃延伸10min,最后16℃保存。改进的CTAB法能成功地提取红叶石楠基因组DNA,利用单因素试验设计所建立的RAPD反应体系,通过重复试验证明了该体系稳定可靠,可应用于红叶石楠遗传多样性、亲缘关系等方面的研究中,为红叶石楠在分子生物学研究奠定了基础。
The reference for the research on the diversity and resource of Photinia faseri and the identification of the relation of its varieties were provided through establish and study the stable system of RAPD reaction.The genomic DNA of Photinia faseri was successfully extracted with the improved CTAB method and studied the concentrations of Mg 2 ＋,dNTPs and primer,and the dosages of template DNA and TaqDNA polymerase which could influence the results of RAPD by the single factor design,established the suitable system of RAPD reaction for Photinia faseri.The results showed that the optimized RAPD reaction conditions were 10×PCR Buffer 2.5μL,Mg 2＋（25mmol/L）2.0μL,primer（20μmol/L）2.0μL,dNTPs（2.5mmol/L）1.8μL,DNA（49.5μg/mL）2.0μL,Taq polymerase 0.3μL（5U/μL） in a total of 25μL reaction system.The appropriate PCR procedure was pre-denaturing at 94℃ for 4min,followed 44 cycles（denaturing at 94℃ for 30s,annealing at 36℃ for 30s,extending at 72℃ for 2min）,extending at 72℃ for 10min and preserving at 16℃.The genomic DNA of Photinia faseri was successfully extracted with the improved CTAB method and the reaction system was established for the procedure of RAPD by the single factor design,through the repeat tests showed that this system was stable and reliable,and could be used in the genetic diversity and genetic relationship of Photinia faseri,it laid the foundation to research Photinia faseri in molecular biology.
出处
《中国农学通报》
CSCD
2012年第34期53-57,共5页
Chinese Agricultural Science Bulletin
基金
江苏省科技厅高新技术研究项目"红叶石楠
三叶草抗逆新品种(系)选育"(BG2007317)
山东省科技厅"山东省良种工程农业生物资源创新利用研究"[鲁农良种字(2010)117号]
